Compounds for the photodecontamination of pathogens in blood

ABSTRACT

Psoralen compound compositions are synthesized which have substitutions on the 4, 4&#39;, 5&#39;, and 8 positions of the psoralen, which yet permit their binding to nucleic acid of pathogens. Reaction conditions that photoactivate these bound psoralens result in covalent crosslinking to nucleic acid, thereby inactivating the pathogen. Higher psoralen binding levels and lower mutagenicity results in safer, more efficient, and reliable inactivation of pathogens. In addition to the psoralen compositions, the invention contemplates inactivating methods using the new psoralens.

FIELD OF THE INVENTION

The present invention provides new psoralens and methods of synthesis ofnew psoralens having enhanced ability to inactivate pathogens in thepresence of ultraviolet light. The present invention also providesmethods of using new and known compounds to inactivate pathogens inhealth related products to be used in vivo and in vitro, and inparticular, blood products.

BACKGROUND

Although improved testing methods for hepatitis B (HBV), hepatitis C(HCV), and human immunodeficiency virus (HIV) have markedly reduced theincidence of transfusion associated diseases, other vital, bacterial,and protozoal agents are not routinely tested for, and remain apotential threat to transfusion safety. Schmunis, G. A., Transfusion31:547-557 (1992). In addition, testing will not insure the safety ofthe blood supply against future unknown pathogens that may enter thedonor population resulting in transfusion associated transmission beforesensitive tests can be implemented.

The recent introduction of a blood test for HCV will reduce transmissionof this virus; however, it has a sensitivity of only 67% for detectionof probable infectious blood units. HCV is responsible for 90% oftransfusion associated hepatitis. Melnick, J. L., abstracts ofVirological Safety Aspects of Plasma, Cannes, November 3-6 (1992) page9. It is estimated that, with the test in place, the risk of infectionis 1 out of 3300 units transfused.

Further, while more sensitive seriological assays are in place for HIV-1and HBV, these agents can nonetheless be transmitted by seronegativeblood donors. International Forum: Vox Sang 32:346 (1977). Ward, J. W.,et al., N. Engl. J. Med., 318:473 (1988). Up to 10% of totaltransfusion-related hepatitis and 25% of severe icteric cases are due tothe HBV transmitted by hepatitis B surface antigen (HBasAg) negativedonors. Vox Sang 32:346(1977). To date, fifteen cases oftransfusion-associated HIV infections have been reported by the Centerfor Disease Control (CDC) among recipients of blood pretested negativefor antibody to HIV-1.

Even if seroconversion tests were a sufficient screen, they may not bepractical in application. For example, CMV (a herpes virus) and parvoB19 virus in humans are common. When they occur in healthy,immunocompetent adults, they nearly always result in asymptomaticseroconversion. Because such a large part of the population isseropositive, exclusion of positive units would result in substantiallimitation of the blood supply.

An alternative approach to eliminate transmission of vital diseasesthrough blood products is to develop a means to inactivate pathogens intransfusion products. Development of an effective technology toinactivate infectious pathogens in blood products offers the potentialto improve the safety of the blood supply, and perhaps to slow theintroduction of new tests, such as the HIV-2 test, for low frequencypathogens. Ultimately, decontamination technology could significantlyreduce the cost of blood products and increase the availability ofscarce blood products. Furthermore, decontamination may extend thestorage life of platelet concentrates which, according to Goldman M. andM. A. Blaichman, Transfusion Medicine Reviews. V: 73-83 (1991), arecurrently limited by potential bacterial contamination.

Several methods have been reported for the inactivation or eliminationof viral agents in erythrocyte-free blood products. Some of thesetechniques, such as heat (Hilfenhous, J., et al., J. Biol. Std. 70:589(1987)), solvent/detergent treatment (Horowitz, B., et al., Transfusion25:516 (1985)), gamma-irradiation (Moroff, G., et al., Transfusion26:453 (1986)), UV radiation combined with beta propriolactone, (PrinceA.M., et al., Reviews of Infect Diseases 5:92-107 (1983)), visible laserlight in combination with hematoporphyrins (Matthews J. L., et al.,Transfusion 28:81-83 (1988); North J., et al., Transfusion 32:121-128(1992)), use of the photoactive dyes aluminum phthalocyananine andmerocyanine 540 (Sieber F., et al., Blood 73:345-350 (1989); Rywkin S.,et al., Blood 78(Suppl 1): 352a (Abstract) (1991)) or UV alone(Proudouz, K. N., et al., Blood 70:589 (1987)) are completelyincompatable with maintainance of platelet function.

Other methods inactivate viral agents by using known furocoumarins, suchas psoralens, in the presence of ultra-violet light. Psoralens aretricyclic compounds formed by the linear fusion of a furan ring with acoumarin. Psoralens can intercalate between the base pairs ofdouble-stranded nucleic acids, forming covalent adducts to pyrimidinebases upon absorption of long wave ultraviolet light (UVA). G. D. Ciminoet al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev.Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to apsoralen-pyrimidine monoadduct and on the opposite strand, absorption ofa second photon can lead to formation of a diadduct which functions asan interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058(1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294(1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S.Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated byreference.

The covalently bonded psoralens act as inhibitors of DNA replication andthus have the potential to stop the replication process. Due to this DNAbinding capability, psoralens are of particular interest in relation tosolving the problems of creating and maintaining a safe blood supply.Some known psoralens have been shown to inactivate viruses in some bloodproducts. H. J. Alter et al., The Lancet (ii:1446) (1988); L. Linet al.,Blood 74:517 (1989) (decontaminating platelet concentrates); G. P.Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, herebyincorporated by reference, describe the use of a combination of8-methoxypsoralen (8-MOP) and irradiation. P. Morel et al., Blood Cells18:27 (1992) show that 300 ug/mL of 8-MOP together with ten hours ofirradiation with ultraviolet light can effectively inactivate viruses inhuman serum. Similar studies using 8-MOP and aminomethyltrimethylpsoralen (AMT) have been reported by other investigators. Dodd R. Y., etal., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et al., ThrombHaemostas 65:1162 (Abstract)(1991). Indeed, the photoinactivation of abroad spectrum of microorganisms has been established, including HBV,HCV, and HIV. [Hanson C. V., Blood Cells: 18:7-24 (1992); Alter, H. J.,et al., The Lancet ii:1446 (1988); Margolis-Nunno H. et al., ThrombHaemostas 65:1162 (Abstract) (1991).]

Psoralen photoinactivation is only feasible if the ability of thepsoralen to inactivate viruses is sufficient to ensure a safety marginin which complete inactivation will occur. On the other hand, thepsoralen must not be such that it will cause damage to blood cells.Previous compounds and protocols have necessitated the removal ofmolecular oxygen from the reaction before exposure to light, to preventdamage to blood products from oxygen radicals produced duringirradiation. See L. Lin et al., Blood 74:517 (1989); U.S. Pat. No.4,727,027, to Wiesehahn. This is a cosily and time consuming procedure.

Finally, some commonly known compounds used in PCD cause undesirablemutagenic effects which appears to increase with increased ability tokill virus. In other words, the more effective the known compounds areat inactivating viruses, the more mutagenic the compounds are, and thus,the less useful they at any point in an inactivation system of productsfor in vivo use.

A new psoralen compound is needed which displays improved ability toinactivate pathogens and low mutagenicity, thereby ensuring safe andcomplete inactivation of pathogens in blood decontamination methods.

SUMMARY OF THE INVENTION

The present invention provides new psoralens and methods of synthesis ofnew psoralens having enhanced ability to inactivate pathogens in thepresence of ultraviolet light which is not linked to mutagenicity. Thepresent invention also provides methods of using new and known compoundsto inactivate pathogens in health related products to be used in vivoand in vitro, and particularly, in blood products and blood products insynthetic media.

With respect to new compounds, the present invention contemplatespsoralen compounds, comprising: a) a substituent R₁ on the 4' carbonatom, selected from the group comprising: --(CH₂)_(u) --NH₂, --(CH₂)_(w)--R₂ --(CH₂)_(z) --NH₂, --(CH₂)_(w) R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z)--NH₂, and --(CH₂)_(w) --R₂ --(CH₂)_(x) R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z)--NH₂ ; wherein R₂, R₃, and R₄ are independently selected from the groupcomprising O and NH, in which u is a whole number from 1 to 10, w is awhole number from 1 and 5, x is a whole number from 2 and 5, y is awhole number from 2 and 5, and z is a whole number from 2 and 6; and b)substituents R₅, R₆, and R₇ on the 4, 5', and 8 carbon atomsrespectively, independently selected from the group comprising H and(CH₂)_(v) CH₃, where v is a whole number from 0 to 5; or a salt thereof.Where an element is "independently selected" from a group, it means thatthe element need not be the same as other elements chosen from the samegroup.

The invention contemplates specific compounds of the above structure,wherein R₁ is --CH₂ --O--(CH₂)₂ --NH₂, and wherein R₅, R₆, and R₇ areall CH₃, wherein R₁ is --CH₂ --O--(CH₂)₂ --O--(CH₂)₂ NH₂, and R₅, R₆,and R₇ are all CH₃, wherein R₁ is --CH₂ --O--(CH₂)₂ --O--(CH₂)₂--NH--(CH₂)₄ --NH₂, and R₅, R₆, and R₇ are all CH₃, wherein R₁ is CH₂--NH--(CH₂)₄ --NH₂, and R₅, R₆, and R₇ are all CH₃, and wherein R₁ isCH₂ --NH--(CH₂)₃ --NH--(CH₂)₄ --NH--(CH₂)₃ --NH₂, and R₅, R₆, and R₇ areall CH₃.

The present invention also contemplates psoralen compounds, comprising:a) a substituent R₁ on the 5' carbon atom, selected from the groupcomprising: --(CH₂)_(u) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂,--(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z) --NH₂, and --(CH₂)_(w)--R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ; wherein R₂,R₃, and R₄ are independently selected from the group comprising O andNH, and in which u is a whole number from 1 to 10, w is a whole numberfrom 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2to 5, and z is a whole number from 2 to 6; and, b) substituents R₅, R₆,and R₇ on the 4, 4', and 8 carbon atoms respectively, independentlyselected from the group comprising H and (CH₂)_(v) CH₃, where v is awhole number from 0 to 5, and where when R₁ is --(CH₂)_(u) --NH₂, R₆ isH; or a salt thereof. The present invention contemplate a specificcompound having the above structure, wherein R₁ is --CH₂ --NH--(CH₂)₄--NH₂, and R₅, R₆ , and R₇ are all CH₃.

The present invention also contemplates psoralen compounds, comprising:a) a substituent R₁ on the 5' carbon atom, selected from the groupcomprising: --(CH₂)_(u) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂,--(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(z) --NH₂, and --(CH₂)_(w)--R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ; wherein R₂,R₃, and R₄ are independently selected from the group comprising O andNH, and in which u is a whole number from 3 to 10, w is a whole numberfrom 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2to 5, and z is a whole number from 2 to 6; and, b) substituents R₅, R₆,and R₇ on the 4, 4', and 8 carbon atoms respectively, independentlyselected from the group comprising H and (CH₂)_(v) CH₃, where v is awhole number from 0 to 5; or a salt thereof. The present inventioncontemplates a specific compound having the above structure, wherein R₁is --CH₂ --NH--(CH₂)₄ --NH₂, and R₅, R₆, and R₇ are all CH₃.

With respect to methods for synthesizing new compounds substituted atthe 4' position of the psoralen, the present invention contemplates amethod of synthesizing 4'-(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen,comprising the steps: a) providing4'-(ω-hydroxy-2-oxa)alkyl-4,5',8-trimethylpsoralen; b) treating4'-(ω-hydroxy-2-oxa)alkyl-4,5',8-trimethylpsoralen with a base andmethanesulfonyl chloride so that4'-(ω-methanesulfonyloxy-2-oxa)alkyl-4,5',8-trimethylpsoralen isproduced; c) treating4'-(ω-methanesulfonyloxy-2-oxa)alkyl-4,5',8-trimethylpsoralen withsodium azide, so that 4'-(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralenis produced, and d) reducing4'-[(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralen so that4'-[(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen is produced.

The present invention further contemplates a method of synthesizing acompound of the structure which has a substituent R₁ on the 4' positionof the psoralen, described above, where R₁ comprises--(CH₂)--O--(CH₂)_(x) --O--(CH₂)_(z) --NH₂, where x=z, comprising thesteps: a) providing a 4'-halomethyl-4,5',8-trimethyl psoralen selectedfrom the group comprising 4'-chloromethyl-4,5',8-trimethyl psoralen,4'-bromomethyl-4,5',8-trimethyl psoralen, and4'-iodomethyl-4,5',8trimethyl psoralen; b) treating said4'-halomethyl-4,5',8-trimethyl psoralen with HO(CH₂)_(x) O(CH₂)_(z) OHso that 4'-(ω-hydroxy-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen isproduced, where n=x+3; c) treating said4'-(ω-hydroxy-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen with a base andmethanesulfonyl chloride so that 4'-(ω-methanesulfonyloxy-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen is produced; d) treating4'-(ω-methanesulfonyloxy-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen withsodium azide so that4'-(ω-azido-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen is produced; and e)reducing 4'-(ω-azido-2,n-dioxa)alkyl4,5',8-trimethylpsoralen so that4'-(ω-amino-2,n-dioxa)alkyl-4,5',8-trimethylpsoralen is produced.

The present invention also contemplates a method of synthesizing4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralen,comprising the steps: a) providing a4'-halomethyl-4,5',8-trimethylpsoralen, selected from the groupcomprising 4'-chloromethyl-4,5',8-trimethyl psoralen,4'-bromomethyl-4,5',8-trimethyl psoralen, and4'-iodomethyl-4,5',8-trimethyl psoralen; b) treating said4'-halomethyl-4,5',8-trimethyl psoralen with diethylene glycol so that4'-(7-hydroxy-2,5-dioxa)heptyl-4,5',8-trimethylpsoralen is produced; c)treating 4'-(7-hydroxy-2,5-dioxa)heptyl-4,5',8-trimethylpsoralen with abase and methanesulfonyl chloride so that4'-(7-methanesulfonyloxy-2,5-dioxa)heptyl-4,5',8-trimethylpsoralen isproduced; d) treating4'(7-methanesulfonyloxy-2,5-dioxa)heptyl-4,5',8-trimethylpsoralen with1, 4-diaminobutane so that4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralen isproduced.

The present invention contemplates a method of synthesizing4'-(ω-amino-2-aza)alkyl-4,5',8-trimethylpsoralen, comprising: a)providing 4'-halomethyl-4,5',8-trimethylpsoralen, selected from thegroup comprising 4'-chloromethyl-4,5',8-trimethyl psoralen,4'-bromomethyl-4,5',8-trimethyl psoralen, and4'-iodomethyl-4,5',8-trimethyl psoralen; b) treating said4'-halomethyl-4,5',8-trimethylpsoralen with 1,ω-aminoalkane to produce4'-(ω-diamino-2-aza)alkyl-4,5',8-trimethylpsoralen.

The present invention additionally contemplates a method of synthesizing4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralen,comprising: a ) providing 4,5',8-trimethylpsoralen-4'-carboxaldehyde; b)treating 4,5',8-trimethylpsoralen-4'-carboxaldehyde with spermine and areducing agent to produce4'-(14-amino-2,6,11-triaza)tetradecane-4,5',8-trimethylpsoralen.

Finally, the present invention contemplates the following method ofsynthesizing 5'-(ω-amino-2-aza)alkyl-4,4',8-trimethylpsoralen,comprising: a) providing a 5'-halomethyl-4,4',8-trimethylpsoralen,selected from the group comprising 5'-chloromethyl-4,4',8-trimethylpsoralen, 5'-bromomethyl-4,4',8-trimethyl psoralen, and5'-iodomethyl-4,4',8-trimethyl psoralen; b) treating said5'-halomethyl-4,4',8-trimethylpsoralen with a 1,ω-diaminoalkane toproduce 5'-(ω-amino-2-aza )alkyl-4,4'8-trimethylpsoralen.

The present invention contemplates methods of inactivatingmicroorganisms in blood preparations, comprising, in the followingorder: a) providing, in any order, i) a compound from the groupcomprising 4'-primaryamino-substituted psoralens and5'-primaryamino-substituted psoralens; ii) photoactivating means forphotoactivating said compounds; and iii) a blood preparation suspectedof being contaminated with a pathogen having nucleic acid; b) addingsaid compound to said blood preparation; and c) photoactivating saidcompound, so as to inactivate said pathogen.

The pathogen can be single cell or multicellular organisms, such asbacteria, fungi, mycoplasma and protozoa, or viruses. The pathogen cancomprise either DNA or RNA, and this nucleic acid can be single strandedor double stranded. In one embodiment, the blood preparation is eitherplatelets or plasma.

The present invention contemplates that the photoactivating meanscomprises a photoactivation device capable of emitting a given intensityof a spectrum of electromagnetic radiation comprising wavelengthsbetween 180 nm and 400 nm, and in particular, between 320 nm and 380 nm.It is preferred that the intensity is between 1 and 30 mW/cm² (e.g.between 10 and 20 mW/cm²) and that the mixture is exposed to thisintensity for between one second and thirty minutes (e.g. ten minutes).

The present invention contemplates embodiments wherein said bloodpreparation is in a synthetic media. In one embodiment, theconcentration of compound is between 0.1 and 250 μM. In a preferredembodiment, the compound is added to said blood preparation at aconcentration of between 10 and 150 μM.

The present invention contemplates embodiments of the methods whereinactivation is performed without limiting the concentration ofmolecular oxygen. Furthermore, there is no need for the use ofcosolvents (e.g. dimethyl sulphoxide (DMSO)) to increase compoundsolubility.

In one embodiment, the present invention contemplates methods ofinactivating microorganisms in blood preparations, wherein the compoundis a 4'-primaryamino-substituted psoralen, comprising: a) a substituentR₁ on the 4' carbon atom, selected from the group comprising:--(CH₂)_(u)--NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(x)--R₃ --(CH₂)_(z) --NH₂, and (CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y)--R₄ --(CH₂)_(z) --NH₂ ; wherein R₂, R₃, and R₄ are independentlyselected from the group comprising O and NH, in which u is a wholenumber from 1 to 10, w is a whole number from 1 to 5, x is a wholenumber from 2 to 5, y is a whole number from 2 to 5, and z is a wholenumber from 2 to 6; and b) substituents R₅, R₆, and R₇ on the 4, 5', and8 carbon atoms respectively, independently selected from the groupcomprising H and (CH₂)_(v) CH₃, where v is a whole number from 0 to 5;or a salt thereof.

Alternatively, the present invention contemplates embodiments of themethod of inactivation, wherein the compound is a5'-primaryamino-substituted psoralen comprising: a) a substituent R₁ onthe 5' carbon atom, selected from the group comprising: --(CH₂)_(u)--NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(x)--R₃ --(CH₂)_(z) --NH₂, and --(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y) --R₄ --(CH₂)_(z) --NH₂ ; wherein R₂, R₃, and R₄ are independentlyselected from the group comprising O and NH, and in which u is a wholenumber from 1 to 10, w is a whole number from 1 to 5, x is a wholenumber from 2 to 5, y is a whole number from 2 to 5, and z is a wholenumber from 2 to 6; and, b) substituents R₅, R₆, and R₇ on the 4, 4',and 8 carbon atoms respectively, independently selected from the groupcomprising H and (CH₂)_(v) CH₃, where v is a whole number from 0 to 5,and where when R₁ is selected from the group comprising --(CH₂)_(u)--NH₂, R₆ is H; or a salt thereof.

Alternatively, the present invention contemplates embodiments of themethod of inactivation, wherein the compound is a5'-primaryamino-substituted psoralen comprising: a) a substituent R₁ onthe 5' carbon atom, selected rrom the group comprising: --(CH₂)_(u) NH₂,--(CH₂)_(w) --R₂ --(CH₂)_(z) --NH₂, --(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃--(CH₂)_(z) --NH₂, and --(CH₂)_(w) --R₂ --(CH₂)_(x) --R₃ --(CH₂)_(y)--R₄ --(CH₂)_(z) --NH₂ ; wherein R₂, R₃, and R₄ are independentlyselected from the group comprising O and NH, and in which u is a wholenumber from 3 to 10, w is a whole number from 1 to 5, x is a wholenumber from 2 to 5, y is a whole number from 2 to 5, and z is a wholenumber from 2 to 6; and, b) substituents R₅, R₆, and R₇ on the 4, 4',and 8 carbon atoms respectively, independently selected from the groupcomprising H and (CH₂)_(v) CH₃, where v is a whole number from 0 to 5;or a salt thereof.

In one embodiment of the method of inactivation, at least two of thecompounds are present. The present invention contemplates embodimentswhere the compound is introduced either in solution, such as water,saline, or a synthetic media, or in a dry formulation. The presentinvention also contemplates that the nucleic acid may be DNA or RNA,single stranded or double stranded.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of the device of thepresent invention.

FIG. 2 is a cross-sectional view of the device shown in FIG. 1 along thelines of 2--2.

FIG. 3 is a cross-sectional view of the device shown in FIG. 1 along thelines of 3--3.

FIG. 4 is a cross-sectional view of the device shown in FIG. 1 along thelines of 4--4.

FIG. 5A is a diagram of the synthesis pathways and chemical structuresof compounds 8, 13, and 14 of the present invention.

FIG. 5B is a diagram of the synthesis pathways and chemical structuresof compounds 2, 4, and 7 of the present invention.

FIG. 5C is a diagram of the synthesis pathways and chemical structuresof compounds 1, 5, 6, 9, and 10 of the present invention.

FIG. 5D is a diagram of the synthesis pathways and chemical structuresof compounds 12 and 15 of the present invention.

FIG. 5E is a diagram of a synthesis pathways and the chemical structureof compound 3 of the present invention.

FIG. 5F is a diagram of a synthesis pathways and the chemical structureof compounds 16 and 17 of the present invention.

FIG. 6 shows the impact of concentration on the log kill of R17 whenCompounds 1-3 of the present invention are photoactivated.

FIG. 7 shows the impact of concentration on the log kill of R17 whenCompounds 3-6 of the present invention are photoactivated.

FIG. 8 shows the impact of concentration on the log kill of R17 whenCompounds 2 and 6 of the present invention are photoactivated.

FIG. 9 shows the impact of concentration on the log kill of R17 whenCompounds 6 and 18 of the present invention are photoactivated.

FIG. 10 shows the impact of concentration on the log kill of R17 whenCompound 16 of the present invention is photoactivated.

FIG. 11 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compound 6 of the present invention.

FIG. 12 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compounds 7, 9 and 10 of the present invention.

FIG. 13 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compounds 7 and 12 of the present invention.

FIG. 14 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compound 15 of the present invention.

FIG. 15 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compound 17 of the present invention.

FIG. 16 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compounds 6 and 17 of the present invention.

FIG. 17 shows the impact of varying Joules of irradiation on the logtiter of R17 for Compounds 6 and 15 of the present invention.

FIG. 18 shows the effect of varying the concentration of Compounds 2 and6 of the present invention, in plasma.

FIG. 19 shows the effect of varying the concentration of Compounds 2 and6 of the present invention, in synthetic medium.

FIG. 20A schematically shows the standard blood product separationapproach used presently in blood banks.

FIG. 20B schematically shows an embodiment of the present inventionwhereby synthetic media is introduced to platelet concentrate preparedas in FIG. 20A.

FIG. 20C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 20B.

DESCRIPTION OF THE INVENTION

The present invention provides new psoralens and methods of synthesis ofnew psoralens having enhanced ability to inactivate pathogens in thepresence of ultraviolet light, which is not linked to mutagenicity. Thenew psoralens are effective against a wide variety of pathogens. Thepresent invention also provides methods of using new and known compoundsto inactivate pathogens in health related products to be used in vivoand in vitro, and in particular, blood products.

The inactivation methods of the present invention provide methods ofinactivating pathogens, and in particular, viruses, in blood productsprior to use in vitro or in vivo. In contrast with previous approaches,the method requires only short irradiation times and there is no need tolimit the concentration of molecular oxygen.

The description of the invention is divided into the following sections:I) Photoactivation Devices, II) Compound Synthesis, III) Binding ofCompounds to Nucleic Acid, IV) Inactivation of Contaminants, and V)Preservation of Biochemical Properties of Material Treated.

I. PHOTOACTIVATION DEVICES

The present invention contemplates devices and methods forphotoactivation and specifically, for photoactivation of photoreactivenucleic acid binding compounds. The present invention contemplatesdevices having an inexpensive source of electromagnetic radiation thatis integrated into a unit. In general, the present inventioncontemplates a photoactivation device for treating photoreactivecompounds, comprising: a) means for providing appropriate wavelengths ofelectromagnetic radiation to cause photoactivation of at least onephotoreactive compound; b) means for supporting a plurality of samplesin a fixed relationship with the radiation providing means duringphotoactivation; and c) means for maintaining the temperature of thesamples within a desired temperature range during photoactivation. Thepresent invention also contemplates methods, comprising: a) supporting aplurality of sample containers, containing one or more photoreactivecompounds, in a fixed relationship with a fluorescent source ofelectromagnetic radiation; b) irradiating the plurality of samplecontainers simultaneously with electromagnetic radiation to causephotoactivation of at least one photoreactive compound; and c)maintaining the temperature of the sample within a desired temperaturerange during photoactivation.

The major features of one embodiment of the device of the presentinvention involve: A) an inexpensive source of ultraviolet radiation ina fixed relationship with the means for supporting the samplecontainers, B) rapid photoactivation, C) large sample processing, D)temperature control of the irradiated samples, and E) inherent safety.

A. Electromagnetic Radiation Source

Many sources of ultraviolet radiation can be successfully used indecontamination protocols with psoralens. For example, some groups haveirradiated sample from above and below by General Electric typeF20T12-BLB fluorescent UVA bulbs with an electric fan blowing gentlyacross the lights to cool the area. Alter, H. J., et al., The Lancet,24:1446 (1988). Another group used Type A405-TLGW/05 long wavelengthultraviolet lamp manufactured by P. W. Allen Co., London placed abovethe virus samples in direct contact with the covers of petri dishescontaining the samples, and was run at room temperature. The totalintensity deliverd to the samples under these conditions was 1.3×10¹⁵photons/sec cm² or 0.7 mW/cm² in the petri dish. Hearst, J. E., andThiry, L., Nucleic Acids Research, 4:1339 (1977). However, withoutintending to be limited to any type of photoactivation device, thepresent invention contemplates several preferred arrangements for thephotoactivation device, as follows.

A preferred photoactivation device of the present invention has aninexpensive source of ultraviolet radiation in a fixed relationship withthe means for supporting the sample vessels. Ultraviolet radiation is aform of energy that occupies a portion of the electromagnetic radiationspectrum (the electromagnetic radiation spectrum ranges from cosmic raysto radio waves). Ultraviolet radiation can come from many natural andartificial sources. Depending on the source of ultraviolet radiation, itmay be accompanied by other (non-ultraviolet) types of electromagneticradiation (e.g. visible light).

Particular types of ultraviolet radiation are herein described in termsof wavelength. Wavelength is herein described in terms of nanometers("nm"; 10⁻⁹ meters). For purposes herein, ultraviolet radiation extendsfrom approximately 180 nm to 400 nm. When a radiation source, by virtueof filters or other means, does not allow radiation below a particularwavelength (e.g. 320 nm), it is said to have a low end "cutoff" at thatwavelength (e.g. "a wavelength cutoff at 300 nanometers"). Similarly,when a radiation source allows only radiation below a particularwavelength (e.g. 360 nm), it is said to have a high end "cutoff" at thatwavelength (e.g. "a wavelength cutoff at 360 nanometers").

For any photochemical reaction it is desired to eliminate or leastminimize any deleterious side reactions. Some of these side reactionscan be caused by the excitation of endogenous chromophores that may bepresent during the photoactivation procedure. In a system where onlynucleic acid and psoralen are present, the endogenous chromophores arethe nucleic acid bases themselves. Restricting the photoactivationprocess to wavelengths greater than 320 nm minimizes direct nucleic aciddamage since there is very little absorption by nucleic acids above 313nm.

In human serum or plasma, for example, the nucleic acid is typicallypresent together with additional biological constituents. If thebiological fluid is just protein, the 320 nm cutoff will be adequate forminimizing side reactions (aromatic amino acids do not absorb above 320nm). If the biological fluid includes other analytes, there may beconstituents that are sensitive to particular wavelengths of light. Inview of the presence of these endogenous constituents, it is intendedthat the device of the present invention be designed to allow forirradiation within a small range of specific and desirable wavelengths,and thus avoid damage blood components. The preferred range of desirablewavelengths is between 320 and 350 nm.

Some selectivity can be achieved by choice of commercial irradiationsources. For example, while typical fluorescent tubes emit wavelengthsranging from 300 nm to above 400 nm (with a broad peak centered around360 nm), BLB type fluorescent lamps are designed to remove wavelengthsabove 400 nm. This, however, only provides an upper end cutoff.

In a preferred embodiment, the device of the present invention comprisesan additional filtering means. In one embodiment, the filtering meanscomprises a glass cut-off filter, such as a piece of Cobalt glass. Inanother embodiment, the filtering means comprises a liquid filtersolution that transmits only a specific region of the electromagneticspectrum, such as an aqueous solution of Co(No3)2. This salt solutionyields a transmission window of 320-400 nm. In a preferred embodiment,the aqueous solution of Co(No3)2 is used in combination with NiSO4 toremove the 365 nm component of the emission spectrum of the fluorescentor arc source employed. The Co--Ni solution preserves its initialtransmission remarkably well even after tens of hours of exposure to thedirect light of high energy sources.

It is not intended that the present invention be limited by theparticular filter employed. Several inorganic salts and glasses satisfythe necessary requirements. For example, cupric sulfate is a most usefulgeneral filter for removing the infrared, when only the ultraviolet isto be isolated. Its stability in intense sources is quite good. Othersalts are known to one skilled in the art. Aperture or reflector lampsmay also be used to achieve specific wavelengths and intensities.

When ultraviolet radiation is herein described in terms of irradiation,it is expressed in terms of intensity flux (milliwatts per squarecentimeter or "mW cm-2"). "Output" is herein defined to encompass boththe emission of radiation (yes or no; on or off) as well as the level ofirradiation. In a preferred embodiment, intensity is monitored at 4locations: 2 for each side of the plane of irradiation.

A preferred source of ultraviolet radiation is a fluorescent source.Fluorescence is a special case of luminescence. Luminescence involvesthe absorption of electromagnetic radiation by a substance and theconversion of the energy into radiation of a different wavelength. Withfluorescence, the substance that is excited by the electromagneticradiation returns to its ground state by emitting a quantum ofelectromagnetic radiation. While fluorescent sources have heretoforebeen thought to be of too low intensity to be useful forphotoactivation, in one embodiment the present invention employsfluorescent sources to achieve results thus far achievable on onlyexpensive equipment.

As used here, fixed relationship is defined as comprising a fixeddistance and geometry between the sample and the light source during thesample irradiation. Distance relates to the distance between the sourceand the sample as it is supported. It is known that light intensity froma point source is inversely related to the square of the distance fromthe point source. Thus, small changes in the distance from the sourcecan have a drastic impact on intensity. Since changes in intensity canimpact photoactivation results, changes in distance are avoided in thedevices of the present invention. This provides reproducibility andrepeatability.

Geometry relates to the positioning of the light source. For example, itcan be imagined that light sources could be placed around the sampleholder in many ways (on the sides, on the bottom, in a circle, etc.).The geometry used in a preferred embodiment of the present inventionallows for uniform light exposure of appropriate intensity for rapidphotoactivation. The geometry of a preferred device of the presentinvention involves multiple sources of linear lamps as opposed to singlepoint sources. In addition, there are several reflective surfaces andseveral absorptive surfaces. Because of this complicated geometry,changes in the location or number of the lamps relative to the positionof the samples to be irradiated are to be avoided in that such changeswill result in intensity changes.

B. Rapid Photoactivation

The light source of the preferred embodiment of the present inventionallows for rapid photoactivation. The intensity characteristics of theirradiation device have been selected to be convenient with theanticipation that many sets of multiple samples may need to beprocessed. With this anticipation, a fifteen minute exposure time orless is a practical goal.

In designing the devices of the present invention, relative position ofthe elements of the preferred device have been optimized to allow forfifteen minutes of irradiation time, so that, when measured for thewavelengths between 320 and 350 nanometers, an intensity flux greaterthan approximately 1 mW cm-2 is provided to the sample vessels.

C. Processing of Large Numbers of Samples As noted, another importantfeature of the photoactivation devices of the present invention is thatthey provide for the processing of large numbers of samples. In thisregard, one element of the devices of the present invention is a meansfor supporting a plurality of sample containers. In the preferredembodiment of the present invention the supporting means comprises atube rack placed between two banks of lights. By accepting commonly usedcommercially available tubes, the device of the present invention allowsfor convenient processing of large numbers of samples.

D. Temperature Control

As noted, one of the important features of the photoactivation devicesof the present invention is temperature control. Temperature control isimportant because the temperature of the sample in the sample at thetime of exposure to light can dramatically impact the results. Forexample, conditions that promote secondary structure in nucleic acidsalso enhance the affinity constants of many psoralen derivatives fornucleic acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). Theseconditions are a mix of both solvent composition and temperature. Withsingle stranded 5S ribosomal RNA, irradiation at low temperaturesenhances the covalent addition of HMT to 5S rRNA by two fold at 4° C.compared to 20° C. Thompson et al., J. Mol. Biol. 147:417 (1981). Evenfurther temperature induced enhancements of psoralen binding have beenreported with synthetic polynucleotides. Thompson et al., Biochemistry21:1363 (1982).

E. Inherent Safety

Ultraviolet radiation can cause severe burns. Depending on the nature ofthe exposure, it may also be carcinogenic. The light source of apreferred embodiment of the present invention is shielded from the user.This is in contrast to the commercial hand-held ultraviolet sources aswell as the large, high intensity sources. In a preferred embodiment,the irradiation source is contained within a housing made of materialthat obstructs the transmission of radiant energy (i.e. an opaquehousing). No irradiation is allowed to pass to the user. This allows forinherent safety for the user.

II. COMPOUND SYNTHESIS

A. Photoactivation Compounds in General

"Photoactivation compounds" (or "photoreactive compounds") defines afamily of compounds that undergo chemical change in response toelectromagnetic radiation. Table 1 is a partial list of photoactivationcompounds.

Table 1. Photoactivation Compounds

Actinomycins

Anthracyclinones

Anthramycin

Benzodipyrones

Fluorenes and fluorenones

Furocoumarins

Mitomycin

Monostral Fast Blue

Norphillin A

Many organic dyes not specifically listed

Phenanthridines

Phenazathionium Salts

Phenazines

Phenothiazines

Phenylazides

Quinolines

Thiaxanthenones

The species of photoreactive compounds described herein is commonlyreferred to as the furocoumarins. In particular, the present inventioncontemplates those compounds described as psoralens:[7H-furo(3,2-g)-(1)-benzopyran-7-one, or β-lactone of6-hydroxy-5-benzofuranacrylic acid], which are linear: ##STR1## and inwhich the two oxygen residues appended to the central aromatic moietyhave a 1, 3 orientation, and further in which the furan ring moiety islinked to the 6 position of the two ring coumarin system. Psoralenderivatives are derived from substitution of the linear furocoumarin atthe 3, 4, 5, 8, 4', or 5'positions.

8-Methoxypsoralen (known in the literature under various named, e.g.,xanthotoxin, methoxsalen, 8-MOP) is a naturally occuring psoralen withrelatively low photoactivated binding to nucleic acids and lowmutagenicity in the Ames assay, described in the following experimentalsection. 4'-Aminomethyl-4,5',8-trimethylpsoralen (AMT) is one of mostreactive nucleic acid binding psoralen derivatives, providing up to 1AMT adduct per 3.5 DNA base pairs. S. T. Isaacs, G. Wiesehahn and L. M.Hallick, NCI Monograph 66:21 (1984). However, AMT also exhibitssignificant levels of mutagenicity. A new group of psoralens was desiredwhich would have the best characteristics of both 8-MOP and AMT: lowmutagenicity and high nucleic acid binding affinity, to ensure safe andthorough inactivation of pathogens. The compounds of the presentinvention were designed to be such compounds.

"4'-primaryamino-substituted psoralens" are defined as psoralencompounds which have an NH₂ group linked to the 4'-position of thepsoralen by a hydrocarbon chain having a total length of 2 to 20carbons, where 0 to 6 of those carbons are independently replaced by NHor O, and each point of replacement is separated from each other pointof replacement by at least two carbons, and is separated from thepsoralen by at least one carbon.

"5'-primaryamino substituted psoralens" are defined as psoralencompounds which have an NH₂ group linked to the 5'-position of thepsoralen by a hydrocarbon chain having a total length of 1 to 20carbons, where 0 to 6 of those carbons are independently replaced by NHor O, and each point of replacement is separated from each other pointof replacement by at least two carbons, and is separated from thepsoralen by at least one carbon.

B. Synthesis of the Psoralens

The present invention contemplates synthesis methods for the novelcompounds of the present invention. Several specific examples of theschemes discussed in this section are shown in FIGS. 5A-5F. For ease ofreference, the compounds in these figures have been numbered fromCompound 1 to Compound 17. For the subclass of the linear psoralens,4,5',8-trialkylpsoralens can be made as follows. The4,8-dialkylcoumarins are prepared from 2-alkylresorcinols and a3-oxoalkanoate ester by the Pechmann reaction (Organic Reactions VolVII, Chap 1, ed. Adams et al, Wiley, N.Y., 1953)). The hydroxy group istreated with an allylating reagent, CH2═CHX--CH(R₈)--Y, where X is ahalide or hydrogen, Y is a halide or sulfonate, and R₈ is H or (CH₂)_(v)CH₃, where v is a whole number from 0 to 4. Claisen rearrangement of theresultant allyl ether gives 4,8-dialkyl-6-allyl-7-hydroxycoumarin. Thecoumarins are converted to the 4,5',8-trialkylpsoralens using one of theprocedures previously described (i.e., see, Bender et al, J. Org. Chem.44:2176 (1979); Kaufman, U.S. Pat. Nos. 4,235,781 and 4,216,154, herebyincorporated by reference). 4,5',8-Trimethylpsoralen is a naturalproduct and is commercially available (Aldrich Chemical Co., Milwaukee,Wis.).

Halomethylation of the 4,5',8-trialkylpsoralens with chloromethyl methylether or bromomethyl methyl ether is described in U.S. Pat. No.4,124,598, to Hearst. Longer chain 4'-(ω-haloalkyl)psoralens (hereinreferred to as 4'-HATP) where alkyl is (CH₂)₂ to (CH₂)₁₀ can be preparedunder Freidel-Crafts conditions as discussed elsewhere (Olah and Kuhn,J. Org. Chem., 1964, 29, 2317; Friedel-Crafts and Related Reactions,Vol. II, Part 2, Olah, ed., Interscience, N.Y., 1964, p. 749). Whilereactions of these halomethyl- intermediates with amines (Hearst et al.,U.S. Pat. No. 4,124,598, and alcohols (Kaufman, U.S. Pat. No. 4,269,852)have been described, there are no literature reports on the formation ofextended chain primary amines, especially those in which the terminalamine is linked to the psoralen by a bridge containing one or moreoxygen or nitrogen atoms. Further, the properties of the lattermaterials, such as decreased mutagenicity are unexpected based on whatis known about previously prepared compounds, such as AMT.

Starting from the 4'-HATP, reaction with an excess of a bis-hydroxycompound, HO--(B)--OH, where B is either an alkyl chain (e.g.,HO--(B)--OH is 1,3-propanediol) or a monoether (e.g., diethylene glycol)or a polyether (e.g., tetraethylene glycol), either neat or with asolvent such as acetone at 20°14 80° C., and a base for the carbonchains longer than halomethyl, gives compound I. ##STR2## The terminalhydroxy group of compound I can be transformed to an amino group under avariety of conditions (for example see Larock, "Comprehensive OrganicTransformations", VCH Publishers, N.Y., 1989). Particularly, the hydroxygroup can be converted to the ester of methanesulfonic acid (structureII). This can subsequently be converted to the azide in refluxingethanol and the azide reduced to the final amine, structure III. Themethod described herein utilizes triphenylphosphine and water in THF forthe reduction but other methods are contemplated.

Conversely, compound II can be reacted with diamines, H2N--(B')--NH2(IV) where B' is an alkyl chain (e.g., 1,4,-butanediamine), a monoether(e.g., 3-oxo-1,5-pentanediamine) or a polyether (e.g.,3,6-dioxa-1,8-octanediamine) to give the final product, compound V. Thisreaction is carried out with an excess of diamine in acetonitrile atreflux, but other solvents and temperatures are equally possible.

It is recognized that alternate preparations for structures III and Vare possible, for example where a linear primary alcohol is preparedwhich already contains the amines in a protected form and subsequentlyreacted with 4'-HATP in the presence of a suitable base.

Some final compounds are desired which contain an NH group in the carbonchain between the primary amino group and the psoralen ring. When thelinkage between this nitrogen and the terminating nitrogen contains onlyCH₂ subunits and oxygen but no other nitrogens (structure VI), theproduct can conveniently be prepared from the (haloalkyl)psoralen andthe appropriate diamine of structure IV. This method is also applicableto final products that contain more than two nitrogens in the chain(structure IX) starting from polyamines of structure VIII (e.g.,norspermidine or spermine [commercially available from Aldrich,Milwaukee, Wis.), however, in this case isomeric structures are alsoformed in considerable amounts. The more preferred method for thepreparation of structure IX is reductive amination of thepsoralen-4'-alkanal (VII) with a polyamine of structure VIII and areducing agent such as sodium cyanoborohydride. This reductive aminationis applicable to the synthesis of compounds VI as well. Thecarboxaldehydes (structure VII, n=0) are known (Isaacs et al, J.Labelled Cmpds. Radiopharm., 1982, 19, 345) and other members of thisgroup can be prepared from the 4'-HATP compounds by conversion of theterminal halo group to an aldehyde functionality (for example, Durst,Adv. Org. Chem. 6:285 (1969)). ##STR3##

Other final products have a terminal amine linked to the psoralen by analkyl chain. These are prepared either by reaction of the 4'-HATP withpotassium phthalimide and subsequent liberation of the desired aminewith hydrazine, or conversion of the 4'-HATP to the cyanide compound,followed by reduction, for example with NaBH₄ --CF₃ CO₂ H. ##STR4##

The discussion of the conversion of 4,5',8-trialkylpsoralens to4'-aminofunctinalized-4,5',8-trialkylpsoralens applies equally well whenthe 4- and/or 8-position is substituted with only a hydrogen, thusproviding 4'-primaryamino-substituted-5', (4 or 8)-dialkylpsoralens and4'-primaryamino-substituted-5'-alkylpsoralens.

The 4,4',8-trialkylpsoralens can be prepared in two steps starting fromthe 4,8-dialkyl-7-hydroxycoumarins discussed above. The coumarin istreated with an α-chloro ketone under basic conditions to give the4,8-dialkyl-7(2-oxoalkoxy)coumarin. Cyclization of this intermediate tothe 4,4',8-trialkylcoumarin occurs by heating in aqueous base. Underidentical conditions to those described above for introducing aprimaryamino-substituted side chain, the 4,4',8-trialkylpsoralens can beconverted to the 5'-(w-haloalkyl)-4,4',8-trialkylpsoralens, (hereincalled 5'-HATP), (Kaufman, U.S. Pat. No. 4,294,822 and U.S. Pat. No.4,298,614). Again, this formation of extended-chain primary amines inwhich the terminal amine is linked to the psoralen by a bridgecontaining one or more oxygen or nitrogen atoms is a novel approach.##STR5##

The discussion of the conversion of 4,4',8-trialkylpsoralens to5'-primaryamino-substituted-4,4',8-trialkylpsoralens applies equallywell when the 4- and/or 8-position is just substituted with a hydrogen,thus providing 5'-primaryamino-substituted-4', (4 or 8)-dialkylpsoralens and 5'-primaryamino-substituted-4'-alkylpsoralens.

Referring back to the synthesis of 4' (or 5')-halomethyl-4, 5' (or4'),8-trialkyl psoralens, the preparation of these criticalintermediates in the synthesis of several compounds presents difficultchallenges. The known method of preparation involves treatment of thestarting psoralen with 50-200 equivalents of highly toxic, and volatilechloromethyl methyl ether or bromomethyl methyl ether. Yields of only30-60% of the desired intermediate are obtained. Described herein, is amuch improved procedure which allows for the synthesis of either isomerof the bromomethyl-trialkylpsoralens by careful control of reactionconditions. ##STR6## Reaction of the 4,8-dialkyl-7-hydroxycoumarin with2-chloro-3-butanone under typical basic conditions, provides4,8-dialkyl-7(1-methyl-2-oxopropyloxy)coumarin (XV). This material isheated in aqueous NaOH to provide 4,8-dialkyl-4', 5'-dimethylpsoralen(XVI). The tetrasubstitutedpsoralen and N-bromosuccinimide are thenrefluxed in a solvent, preferably with a catalyst such as benzoylperoxide. If the solvent used is carbon tetrachloride, 4,8-dialkyl-5'bromomethyl4'-methylpsoralen (XVIII) is obtained m greater than 66%Yield. If methylene chloride is used, only4,8-dialkyl-4'-bromomethyl-5'-methylpsoralen (XVII) is obtained in ≧80%yield. Benzylic bromination in other solvents can also be done,generating one of the isomeric products alone or in a mixture. Thesesolvents include, but are not limited to, chloroform,bromotrichloromethane and benzene.

The discussion above of the syntheses of 4'-primaryamino- and5'-primaryamino-psoralens can be extended to the non-linear coumarins,specifically the isopsoralens or angelicins. Thus, the4'-chloromethylangelicins (IXX) and the 5'-chloromethylangelicins (XX)can be prepared in a similar manner to their linear counterparts. Byanalogy with the synthetic pathways presented above one can envision thesynthesis of 4'(w-amino)alkylangelicins and 5'-(ω-amino)alkylangelicinswhere the alkyl linkage can contain one or more oxygen or nitrogenatoms. ##STR7## III. BINDING OF COMPOUNDS TO NUCLEIC ACID

The present invention contemplates binding new and known compounds tonucleic acid, including (but not limited to) viral nucleic acid andbacterial nucleic acid. One approach of the present invention to bindingphotoactivation compounds to nucleic acid is photobinding. Photobindingis defined as the binding of photobinding compounds in the presence ofphotoactivating wavelengths of light. Photobinding compounds arecompounds that bind to nucleic acid in the presence of photoactivatingwavelengths of light. The present invention contemplates methods ofphotobinding with photobinding compounds of the present invention.

One embodiment of the method of the present invention for photobindinginvolves the steps: a) providing a photobinding compound of the presentinvention; and b) mixing the photobinding compound with nucleic acid inthe presence of photoactivation wavelengths of electromagneticradiation.

The invention further contemplates a method for modifying nucleic acid,comprising the steps: a) providing photobinding compound of the presentinvention and nucleic acid; and b) photobinding the photobindingcompound to the nucleic acid, so that a compound:nucleic acid complex isformed.

IV. INACTIVATION OF PATHOGENS

The present invention contemplates treating a blood product with aphotoactivation compound and irradiating to inactivate contaminatingpathogen nucleic acid sequences before using the blood product.

A. Inactivation In General

The term "inactivation" is here defined as the altering of the nucleicacid of a unit of pathogen so as to render the unit of pathogenincapable of replication. This is distinct from "total inactivation",where all pathogen units present in a given sample are renderedincapable of replication, or "substantial inactivation," where most ofthe pathogen units present are rendered incapable of replication."Inactivation efficiency" of a compound is defined as the level ofinactivation the compound can achieve at a given concentration ofcompound or dose of irradiation. For example, if 100 μM of ahypothetical compound X inactivated 5 logs of HIV virus whereas underthe same experimental conditions, the same concentration of compound Yinactivated only 1 log of virus, then compound X would have a better"inactivation efficiency" than compound Y.

To appreciate that an "inactivation" method may or may not achieve"total inactivation," it is useful to consider a specific example. Abacterial culture is said to be inactivated if an aliquot of theculture, when transferred to a fresh culture plate and permitted togrow, is undetectable after a certain time period. A minimal number ofviable bacteria must be applied to the plate for a signal to bedetectable. With the optimum detection method, this minimal number is 1bacterial cell. With a suboptimal detection method, the minimal numberof bacterial cells applied so that a signal is observed may be muchgreater than 1. The detection method determines a "threshold" belowwhich the "inactivation method" appears to be completely effective (andabove which "inactivation" is, in fact, only partially effective).

B. Inactivation of Potential Pathogens

The same considerations of detection method and threshold are presentwhen determining the sensitivity limit of an inactivation method fornucleic acid. Again, by "inactivation" it is meant that a unit ofpathogen is rendered incapable of replication.

In the case of inactivation methods for material to be used by humans,whether in vivo or in vitro, the detection method can theoretically betaken to be the measurement of the level of infection with a disease asa result of exposure to the material. The threshold below which theinactivation method is complete is then taken to be the level ofinactivation which is sufficient to prevent disease from occuring due tocontact with the material. It is recognized that in this practicalscenario, it is not essential that the methods of the present inventionresult in "total inactivation". That is to say, "substantialinactivation" will be adequate as long as the viable portion isinsufficient to cause disease. The inactivation method of the presentinvention renders nucleic acid in pathogens substantially inactivated.In one embodiment, the inactivation method renders pathogen nucleic acidin blood preparations substantially inactivated.

Without intending to be limited to any method by which the compounds ofthe present invention inactivate pathogens, it is believed thatinactivation results from light induced binding of psoralens to pathogennucleic acid. Further, while it is not intended that the inactivationmethod of the present invention be limited by the nature of the nucleicacid; it is contemplated that the inactivation method render all formsof nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.

In the case of photoactivation compounds modifying nucleic acid, it ispreferred that interaction of the pathogen nucleic acid (whether DNA,mRNA, etc.) with the photoactivation compound causes the pathogen to beunable to replicate, such that, should a human be exposed to the treatedpathogen, infection will not result.

"Synthetic media" is herein defined as an aqueous synthetic blood orblood product storage media. In one embodiment, the present inventioncontemplates inactivating blood products in synthetic media. This methodreduces harm to blood products and permits the use of much lowerconcentrations of photoactivation compounds.

The psoralen photoinactivation method inactivates nucleic acid basedpathogens present in blood through a single procedure. Thus, it has thepotential to eliminate bacteria, protozoa, and viruses as well. Had aneffective decontamination method been available prior to the advent ofthe AIDS pandemic, no transfusion associated HIV transmission would haveoccurred. Psoralen-based decontamination has the potential to eliminateall infectious agents from the blood supply, regardless of the pathogeninvolved. Additionally, psoralen-based decontamination has the abilityto sterilize blood products after

                  TABLE 2                                                         ______________________________________                                        Viruses Photochemically Inactivated by Psoralens                              Family          Virus                                                         ______________________________________                                        Adeno           Adenovirus 2                                                                  Canine hepatitis                                              Arena           Pichinde                                                                      Lassa                                                         Bunya           Turlock                                                                       California encephalitis                                       Herpes          Herpes simplex 1                                                              herpes simplex 2                                                              Cytomegalovirus                                                               Pseudorabies                                                  Orothomyxo      Influenza                                                     Papova          SV-40                                                         Paramyxo        Measles                                                                       Mumps                                                                         Parainfluenza 2 and 3                                         Picorna.sup.1   Poliovirus 1 and 2                                                            Coxsackie A-9                                                                 Echo 11                                                       Pox             Vaccinia                                                                      Fowl Pox                                                      Reo             Reovirus 3                                                                    Blue tongue                                                                   Colorado tick fever                                           Retro           HIV                                                                           Avian sarcoma                                                                 Murine sarcome                                                                Murine leukemia                                               Rhabdo          Vesticular stomatitis virus                                   Toga            Western equine encephalitis                                                   Dengue 2                                                                      Dengue 4                                                                      St. Louis encephalitis                                        Hepadna         hepatitis B                                                   Bacteriophage   Lambda                                                                        T2                                                            (Rickettsia)    R. akari (rickettsialpox)                                     ______________________________________                                         .sup.1 In the article, it was pointed out that Piconaviruses were             photoinactivated only if psoralens were present during virus growth.     

collection and processing, which in the case of platelet concentratescould solve the problem of low level bacterial contamination and resultin extended storage life. Morrow J. F., et al., JAMA 266:555-558 (1991);Bertolini F., et al., Transfusion 32:152-156 (1992).

A list of viruses which have been photochemically inactivated by one ormore psoralen derivatives appears in Table 2. (From Table 1 of Hanson,C. V., Blood Cells 18:7 (1992)). This list is not exhaustive, and ismerely representative of the great variety of pathogens psoralens caninactivate. The present invention contemplates the inactivation of theseand other viruses by the compounds described herein. The compounds ofthe present invention are particularly well suited for inactivatingenvelope viruses, such as the HIV virus.

C. Selecting Photoctivation Compounds for Inactivation of Pathogens

In order to evaluate a compound to decide if it would be useful in themethods of the present invention, two important properties should beconsidered: the compound's ability to inactivate pathogens and itsmutagenicity. The ability of a compound to inactivate pathogens may bedetermined by several methods. One technique is to perform abacteriophage screen; an assay which determines nucleic acid binding oftest compounds. A screen of this type, an R17 screen, is described indetail in EXAMPLE 9, below. Another technique is to perform a viralscreen, as shown in detail in EXAMPLE 10 for HIV, and EXAMPLE 11 forDuck Hepatitis B Virus. The R17 bacteriophage screen is believed to bepredictive of HIV inactivation efficiency, as well as the efficiency ofcompounds against many other viruses. R17 was chosen because it wasexpected to be a very difficult pathogen to inactivate. It is a small,single stranded RNA phage. Without intending to be limited to any meansby which the present invention operates, it is expected that shorterpieces of nucleic acid are harder to inactivate because they require ahigher frequency of formation of psoralen adducts than do longer piecesof nucleic acid. Further, single stranded RNA pathogens are moredifficult to inactivate because psoralens can neither intercalatebetween base pairs, as with double-stranded nucleic acids, nor formdiadducts which function as interstrand crosslinks. Thus it is expectedthat when inactivation of R17 is achieved, these same conditions willcause the inactivation of many viruses and bacteria.

The second property that is important in testing a compound for use inmethods of the present invention is mutagenicity. The most widely usedmutagen/carcinogen screening assay is the Ames test. This assay isdescribed by D. M. Maron and B. N. Ames in Mutation Research 113:173(1983). The Ames test utilizes several unique strains of Salmonellatyphimurium that are histidine-dependent for growth and that lack theusual DNA repair enzymes. The frequency of normal mutations that renderthe bacteria independent of histidine (i.e., the frequency ofspontaneous revertants) is low. Thus, the test can evaluate the impactof a compound on this revertant frequency.

Because some substances are not mutagenic by themselves, but areconverted to a mutagen by metabolic action, the compound to be tested ismixed with the bacteria on agar plates along with the liver extract. Theliver extract serves to mimic metabolic action in an animal. Controlplates have only the bacteria and the extract.

The mixtures are allowed to incubate. Growth of bacteria (if any) ischecked by counting colonies. A positive Ames test is one where thenumber of colonies on the plates with mixtures containing the compoundsignificantly exceeds the number on the corresponding control plates.

When known carcinogens are screened in this manner with the Ames test,approximately ninety percent are positive. When known noncarcinogens aresimilarly tested, approximately ninety percent are negative. Byperforming these screens, a person skilled in the art can quicklydetermine which compounds would be appropriate for use in methods of thepresent invention.

D. Delivery of Compounds for Photoinactivation

The present invention contemplates several different formulations androutes by which the compounds described herein can be delivered in aninactivation method. This section is merely illustrative, and notintended to limit the invention to any form or method of introducing thecompound.

The compounds of the present invention may be introduced in aninactivation method in several forms. The compounds may be introduced asan aqueous solution in water, saline, a synthetic media such as"Sterilyte™, or a variety of other solvents. The compounds can furtherbe provided as dry formulations, with or without adjuvants.

The new compounds may also be provided by many different routes. Forexample, the compound may be introduced to the reaction vessel, such asa blood bag, at the point of manufacture. Alternatively, the compoundmay be added to the material to be sterilized after the material hasbeen placed in the reaction vessel. Further, the compounds may beintroduced alone, or in a "cocktail" or mixture of several differentcompounds.

V. PRESERVATION OF BIOCHEMICAL PROPERTIES OF MATERIAL TREATED

Psoralens are useful in inactivation procedures, because the reactioncan be carried out at temperatures compatible with retaining biochemicalproperties of blood and blood products. Hanson, C. V., Blood Cells 18:7(1992). The inactivation compounds and methods of the present inventionare especially useful because they display the unlinking of pathogeninactivation efficiency from mutagenicity. The compounds exhibitpowerful pathogenic inactivation without a concomitant rise inmutagenicity. The commonly known compounds tested in photoinactivationprotocols, such as AMT, appear to exhibit a link between pathogeninactivation efficiency and mutagenetic action that until now seemedindivisible.

While it is not intended that the present invention be limited to anytheory by which pathogen inactivation efficiency is unlinked frommutagenicity, it is postulated that unlinking occurs as a result of thelength of the groups substituted on the psoralen, and the location ofcharges on the compounds. It is postulated that positive charges on oneor both ends of mutagenic compounds have non-covalent interactions withthe phosphate backbone of DNA. These interactions are presumed to occurindependent of the presence of light (called "dark binding"). In theory,the psoralen thereby sterically blocks polymerase from opening up theDNA, causing mutagenicity. In contrast, compounds of the presentinvention carry a positive or neutral charge on a long substitute group.These substituted groups form a steric barrier during dark binding thatis much easier to free from the DNA, permitting polymerase to pass. Thusno mutagenicity results.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); Kg(kilograms); L (liters); mL (milliliters); μL(microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); J(Joules, note that in FIGS. 6, 8-17, Joules or J refers to Joules/cm²);°C. (degrees Centigrade); TLC (Thin Layer Chromatography); EAA(ethylacetoacetate); EtOH (ethanol); HOAc (acetic acid); W (watts); mW(milliwatts); NMR (Nuclear Magnetic Resonance; spectra obtained at roomtemperature on a Varian Gemini 200 MHz Fourier Transform Spectrometer);m.p. (melting point); UV (ultraviolet light); THF (tetrahydrofuran);DMEM (Dulbecco's Modified Eagles Medium); FBS (fetal bovine serum); LB(Luria Broth); EDTA (ethelene diamine tetracidic acid).

For ease of reference, some compounds of the present invention have beenassigned a number from 1-17. The reference numbers are assigned in FIGS.5A-5F and appear below the structure of each compound. These referencenumbers are used throughout the experimental section.

When isolating compounds of the present invention in the form of an acidaddition salt, the acid is preferably selected so as to contain an anionwhich is non-toxic and pharmacologically acceptable, at least in usualtherapeutic doses. Representative salts which are included in thispreferred group are the hydrochlorides, hydrobromides, sulphates,acetates, phosphates, nitrates, methanesulphonates, ethanesulphonates,lactates, citrates, tartrates or bitartrates, and maleates. Other acidsare likewise suitable and may be employed as desired. For example,fumaric, benzoic, ascorbic, succinic, salicylic, bismethylenesalicylic,propionic, gluconic, malic, malonic, mandelic, cinnamic, citraconic,stearic, palmitic, iraconic, glycolic, benzenesulphonic, and sulphamicacids may also be employed as acid addition salt-forming acids.

In one of the examples below, phosphate buffered synthetic media isformulated for platelet treatment. This can be formulated in one step,resulting in a pH balanced solution (e.g. pH 7.2), by combining thefollowing reagents in 2 liters of distilled water:

    ______________________________________                                        Preparation of Sterilyte ™ 3.0                                                       Formula W.                                                                             mMolarity Grams/2 Liters                                   ______________________________________                                        NaAcetate*3H.sub.2 O                                                                      136.08     20        5.443                                        Glucose     180.16      2        0.721                                        D-mannitol  182.17     20        7.287                                        KCl          74.56      4        0.596                                        NaCl         58.44     100       11.688                                       Na.sub.3 Citrate                                                                          294.10     10        5.882                                        Na.sub.2 HPO.sub.4 *7H.sub.2 O                                                            268.07       14.46   7.752                                        NaH.sub.2 PO.sub.4 *H.sub.2 O                                                             137.99        5.54   1.529                                        MgCl.sub.2 *6H.sub.2 O                                                                    203.3       2        0.813                                        ______________________________________                                    

The solution is then mixed, sterile filtered (0.2 micron filter) andrefrigerated.

The Polymerase Chain Reaction (PCR) is used in one of the examples tomeasure whether viral inactivation by some compounds was complete. PCRis a method for increasing the concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202, herebyincorporated by reference. This process for amplifying the targetsequence consists of introducing a large excess of two oligonucleotideprimers to the DNA mixture containing the desired target sequence,followed by a precise sequence of thermal cycling in the presence of aDNA polymerase. The two primers are complementary to their respectivestrands of the double stranded target sequence. To effect amplification,the mixture is denatured and the primers then to annealed to theircomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing, andpolymerase extension can be repeated many times (i.e. denaturation,annealing and extension constitute one "cycle;" there can be numerous"cycles") to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to by the inventors as the "PolymeraseChain Reaction". Because the desired amplified segments of the targetsequence become the predominant sequences (in terms of concentration) inthe mixture, they are said to be "PCR amplified".

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g. hybridization with a labelled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³² P labelled deoxynucleotide triphosphates, e.g. dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules.

The PCR amplification process is known to reach a plateau concentrationof specific target sequences of approximately 10⁻⁸ M. A typical reactionvolume is 100 μl, which corresponds to a yield of 6×10¹¹ double strandedproduct molecules.

PCR is a polynucleotide amplification protocol. The amplification factorthat is observed is related to the number (n) of cycles of PCR that haveoccurred and the efficiency of replication at each cycle (E), which inturn is a function of the priming and extension efficiencies during eachcycle. Amplification has been observed to follow the form E^(n), untilhigh concentrations of PCR product are made. At these highconcentrations (approximately 10⁻⁸ M/l) the efficiency of replicationfalls off drastically. This is probably due to the displacement of theshort oligonucleotide primers by the longer complementary strands of PCRproduct. At concentrations in excess of 10⁻⁸ M, the rate of the twocomplementary PCR amplified product strands finding each other duringthe priming reactions become sufficiently fast that this occurs beforeor concomitant with the extension step of the PCR procedure. Thisultimately leads to a reduced priming efficiency, and therefore, areduced cycle efficiency. Continued cycles of PCR lead to decliningincreases of PCR product molecules. PCR product eventually reaches aplateau concentration.

The sequences of the polynucleotide primers used in this experimentalsection are as follows:

DCD03:5'ACT AGA AAA CCT CGT GGA CT 3'

DCD05:5'GGG AGA GGG GAG CCC GCA CG 3'

DCD06:5'CAA TTT CGG GAA GGG CAC TC 3'

DCD07:5'GCT AGT ATT CCC CCG AAG GT 3'

With DCD03 as a common forward primer, the pairs generate amplicons oflength 127, 327, and 1072 bp. These oligos were selected from regionsthat are absolutely conserved between 5 different dHBV isolates (DHBV1,DHBV3, DHBV16, DHBV22, and DHBV26) as well as from heron HBV (HHBV4).

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1

As noted above, the present invention contemplates devices and methodsfor the photoactivation of photoreactive nucleic acid binding compounds.In this example, a photoactivation device is described fordecontaminating blood products according to the method of the presentinvention. This device comprises: a) means for providing appropriatewavelengths of electromagnetic radiation to cause photoactivation of atleast one photoreactive compound; b) means for supporting a plurality ofblood products in a fixed relationship with the radiation providingmeans during photoactivation; and c) means for maintaining thetemperature of the blood products within a desired temperature rangeduring photoactivation.

FIG. 1 is a perspective view of one embodiment of the device integratingthe above-named features. The figure shows an opaque housing (100) witha portion of it removed, containing an array of bulbs (101) above andbelow a plurality of representative blood product containing means (102)placed between plate assemblies (103, 104). The plate assemblies (103,104) are described more fully, subsequently.

The bulbs (101), which are connectable to a power source (not shown),serve as a source of electromagnetic radiation. While not limited to theparticular bulb type, the embodiment is configured to accept an industrystandard, dual bipin lamp.

The housing (100) can be opened via a latch (105) so that the bloodproduct can be placed appropriately. As shown in FIG. 1, the housing(100), when closed, completely contains the irradiation from the bulbs(101). During irradiation, the user can confirm that the device isoperating by looking through a safety viewport (106) which does notallow transmission of ultraviolet light to the user.

The housing (100) also serves as a mount for several electroniccomponents on a control board (107), including, by way of example, amain power switch, a count down timer, and an hour meter. Forconvenience, the power switch can be wired to the count down timer whichin turn is wired in parallel to an hour meter and to the source of theelectromagnetic radiation. The count down timer permits a user to presetthe irradiation time to a desired level of exposure. The hour metermaintains a record of the total number of radiation hours that areprovided by the source of electromagnetic radiation. This featurepermits the bulbs (101) to be monitored and changed before their outputdiminishes below a minimum level necessary for rapid photoactivation.

FIG. 2 is a cross-sectional view of the device shown in FIG. 1 along thelines of 2--2. FIG. 2 shows the arrangement of the bulbs (101) with thehousing (100) opened. A reflector (108A, 108B) completely surrounds eacharray of bulbs (101). Blood product containing means (102) are placedbetween upper (103) and lower (104) plate assemblies. Each plateassembly is comprised of an upper (103A, 104A) and lower (103B, 104B)plates. The plate assemblies (103, 104) are connected via a hinge (109)which is designed to accommodate the space created by the blood productcontaining means (102). The upper plate assembly (103) is brought torest gently on top of the blood product containing means (102) supportedby the lower plate (104B) of the lower plate assembly (104).

Detectors (110A, 110B, 110C, 110D) may be conveniently placed betweenthe plates (103A, 103B, 104A, 104B) of the plate assemblies (103, 104).They can be wired to a printed circuit board (111) which in turn iswired to the control board

FIG. 3 is a cross-sectional view of the device shown in FIG. 1 along thelines of 3--3. Six blood product containing means (102) (e.g. Teflon™platelet unit bags) are placed in a fixed relationship above an array ofbulbs (101). The temperature of the blood product can be controlled viaa fan (112) alone or, more preferably, by employing a heat exchanger(113) having cooling inlet (114) and outlet (115) ports connected to acooling source (not shown).

FIG. 4 is a cross-sectional view of the device shown in FIG. 1 along thelines of 4--4. FIG. 4 more clearly shows the temperature controlapproach of a preferred embodiment of the device. Upper plate assemblyplates (103A, 103B) and lower plate assembly plates (104A, 104B) eachcreate a temperature control chamber (103C, 104C), respectively. The fan(112) can circulate air within and between the chambers (103C, 104C).When the heat exchanger (113) is employed, the circulating air is cooledand passed between the plates (103A, 103B, 104A, 104B).

EXAMPLE 2

Synthesis of 4'(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralenHydrochloride (Compound 2) and Related Compounds (Compound 4)

The preparation of 4'-chloromethyl-4,5',8-trimethylpsoralen fromcommercially available 4,5',8-trimethylpsoralen has been previouslydescribed (U.S. Pat. No. 4,124,598; Isaacs et al., Biochem. 16:1058(1977)). Reaction of the chloromethyl compound with alcohols (U.S. Pat.No. 4,124,598), pyridine (U.S. Pat. No. 4,169,204), glycol andaminoethanol (U.S. Pat. No. 4,269,852) have all been previouslyreported. However, compounds in which the 4'-position is substitutedwith a group, CH₂ --X--NH₂, where X=alkyl or (poly)aza- or oxaalkyl havenot been described. The synthesis of4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen hydrochloride isachieved in four (4) steps:

STEP 1:4'-Chloromethyl-4,5',8-trimethylpsoralen (550 mg, 1.99 mmol) andethylene glycol (6.8 ml, 121.9 mmol) were heated in acetone (6 mL) to50°-60° C. for 3.5 hrs. After 2 hrs heating, the white suspension hadturned to a clear light yellow solution. The acetone and ethylene glycolwere removed on the rotoevaporator and water (50 mL) was added to theresidue. The resultant suspension was filtered, washed with cold waterthen dried in the vacuum oven to give 574 mg (96%) of4'-(4-hydroxy-2-oxa)butyl-4,5',8-trimethylpsoralen; NMR (CDCl₃) δ: 2.51(s, 6H); 2.58 (s, 3H); 3.62 (t, J=4.5 Hz, 2H); 3.78 (t, J=4.9 Hz, 2H);4.70 (s, 2H); 6.26 (d, J=1.1 Hz, 1H); 7.61 (s, 1H).

STEP 2: 4'(4-hydroxy-2-oxa)butyl-4,5',8-trimethylpsoralen (574 mg, 1.9mmol) was dissolved in CH₂ Cl₂ (6 mL) under N₂ at ≦10° C. Triethylamne(359 mg, 3.55 mmol) was added. Methanesulfonyl chloride (305 mg, 266mmol) was dropped in slowly keeping the temperature below 10° C. Afteraddition was completed the mixture was stirred for 15 more minutes andthen it was stirred at room temperature for 10 hours. To the reactedsuspension CH₂ Cl₂ (45 mL) was added and the mixture was washed withwater (20×3 mL), then dried over anhydrous Na₂ SO₄. Concentration at≦30° C. followed by vacuum drying gave4'-[(4-methanesulfonyloxy-2-oxa)butyl-4,5'.8-trimethylpsoralen as ayellow solid (706 mg, 98%), mp 138°-140° C. NMR δ2.51 (s, 3H);2.52 (d, 3H);2.58 (s, 3H);2.99 (s, 3H); 3.77 (m ,2H); 4.39 (m, 2H); 4.71 (s, 2H);6.26(s, 1H); 7.62 (s, 1H).

STEP 3:4'-[(4-Methanesulfonyloxy-2-oxa)butyl-4,5',8-trimethylpsoralen(706 mg, 1.86 mmol) and sodium azide (241 mg, 3.71 mmol) were refluxedin 95% ethyl alcohol (5 mL) for 8 hours. The reaction solution wascooled and cold water (55 mL) was added. The off-white solid wasfiltered and washed with cold water. Upon vacuum drying, the azide wasobtained as a light yellowish solid (575 mg, 95% mp 105°-106° C. NMRδ2.51 (s, 6H); 2.58 (s, 3H); 3.41 (t, J=4.9 Hz, 2H); 3.67 (apparent t,J=4.9 Hz, 2H); 4.70 (s, 2H); 6.26 (s, 1H); 7.66 (s, 1H).

STEP 4:4'-(4-Azido-2-oxa)butyl-4,5',8-trimethylpsoralen (1.65 g, 5.03mmol) was dissolved in tetrahydrofuran (10 mL). Triphenylphospine (1.59g, 6.08 mmol) and six drops of water were added to the foregoingsolution. After stirring at room temperature overnight, the light yellowsolution was concentrated. The residue was dissolved in CHCl₃ (90 mL)and extracted with 0.3N aqueous HCl (30 mL, then 2×5 mL). Combined HCllayers was carefully treated with K₂ CO₃ until saturated. The basesolution was extracted with CHCl₃ (3×60 mL). Combined CHCl₃ layers werewashed with 60 mL of water, 60 mL of brine and dried over anhydrous Na₂SO₄. Upon concentration and vacuum drying the amine was obtained as ayellow solid (1.25 g, 82%), mp 139°-141° C.; NMR δ2.48 (s, 6H); 2.55 (s,3H); 2.89 (t, J=6 Hz, 2H); 3.52 (t, J=6 Hz, 2H); 4.64 (s, 2H);6.22 (s,1H);7.59 (s, 1H).

The amine was dissolved in absolute ethanol (40 mL) and 20 mL of 1N HClin ethyl ether was added. After sitting at 5° C. overnight, theprecipitate was filtered and rinsed with ether to give 1.25 g ofCompound 2, mp 236° C. (decomp). Anal. Calculated for C₁₇ H₂₀ ClNO₄ : C,60.45: H,5.97; N, 4.15. Found: C, 60.27; H, 5.88; N, 4.10.

Similarly prepared was4'-(5-amino-2-oxa)pentyl-4,5',8-trimethylpsoralen, (Compound 4), m.p.212°-214° C. (decomposed). NMR of the free base: δ1.73 (pent, J=6.4 Hz,2H),2.45(s, 6H),2.51 (s, 3H),2.78 (t,J=6.8 Hz, 2H), 3.54 (t, J6.2 Hz,2H),4.59 (s, 2H),6.18 (s, 1H),7.54 (s, 1H).

EXAMPLE 3 Synthesis of4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralen Hydrochloride(Compound 7)

The synthesis of 4'-(7-amino-2,5-oxa)heptyl-4,5',8-trimethylpsoralenhydrochloride proceeds in four (4) steps:

STEP 1:4'-Chloromethyl-4,5',8-trimethylpsoralen (589 mg, 2.13 mmol),diethylene glycol (15.4 g, 145 mmol) and acetone (13 mL) were refluxedfor 11.5 hours. The reaction solution was concentrated to remove acetoneand part of the diethylene glycol. To the resulting light brown solutionwas added CHCl₃ (40 mL), then washed with water several times. The CHCl₃layer was dried over anhydrous Na₂ SO₄ and concentrated to give 781 mgof product (˜100%). NMR δ2.46 (d, 3H),2.47 (s, 3H),2.51 (s, 3H),3.58-3.67 (m, 8H), 4.67 (s, 2H), 6.18 (s, 1H),7.57 (s, 1H).

STEP 2:4'-(7-Hydroxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (781 mg,2.25 mmol) was dissolved in CH₂ Cl₂ (2.5 mL) under a N₂ stream at <10°C. Triethylamine (363 mg, 3.59 mmol) was added. Methanesulfonyl chloride(362 mg, 3.16 mmol) was slowly dropped in to keep the temperature below10° C. After addition was completed, the mixture was kept below 10° C.for 15 more minutes. The mixture was stirred at room temperatureovernight then CH₂ Cl₂ (50 mL) was added. The solution was washed withwater (3×60 mL), dried over anhydrous Na₂ SO₄ and concentrated at ≦30°C. Upon vacuum drying, a light brown syrup was obtained; 437 mg (76%).NMR δ2.50 (s, 3H),2.51 (s, 3H),2.58 (s, 3H),3.01 (s, 3H), 3.66 (m,4H),3.77 (t, J=4.6 Hz, 2H),4.37 (t, J=6 Hz, 2H), 4.69 (s, 2H),6.25 (s,1H),7.61 (s, 1H)

STEP 3:4'-(7-Methanesulfonyloxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen(288 mg, 0.678 mmol) and sodium azide (88.2 mg, 1.36 mmol) were refluxedin 3 mL of 95% ethyl alcohol for 8 hours. The reaction solution was letcool and cold water (50 mL) was added. The water layer was poured away.The crude material was purified by chromatography on (Silica gel withchloroform eluent) a Chromatotron (Harrison Research, Inc., Palo Alto,Calif.) and vacuum dried to give a light yellow syrup, (123 mg, 49%).NMR δ2.50 (s, 6H),2.57 (s, 3H),3.39 (t, J=5.2 Hz, 2H),3.68 (m, 6H),4.70(s, 2H), 6.24 (s, 1H),7.62 (s, 1H)

STEP 4:4'-(7-Azido-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (122 mg, 0.33mmol), triphenylphosphine (129 mg, 0.49 mmol) and several drops of waterwere dissolved in tetrahydrofuran (2 mL). The light yellow clearsolution was stirred at room temperature over a weekend; no startingmaterial was detected by TLC. The reaction solution was concentrated andthe residue was dissolved in CHCl₃ (20 mL). The solution was extractedwith 0.15N aqueous HCl solution (10 mL then 2×5 mL) and the HCl layerswas taken to pH 13 by addition of 20% aqueous NaOH solution. The basicsolution was extracted with CHCl₃ (3×15 mL). The combined CHCl₃ layerswere washed with water, dried over anhydrous Na₂ SO₄ concentrated, andvacuum dried to give 63.9 mg of product (56%). TLC showed only one spot.NMR δ2.50 (s, 3H); 2.50 (s, 3H);2.57 (s, 3H);2.86 (t, J=5.3 Hz, 2H);3.50 (t, J=5.3 Hz, 2H); 3.63 (s, 4H); 4.70 (s, 2H); 6.24 (s, 1H); 7.62(s, 1H). m.p. 170°-173° C.

The solid was dissolved in absolute ethanol, then 1M HCl in ethyl etherwas added, the suspension was filtered and the product rinsed with etherand dried.

EXAMPLE 4 Synthesis of4'-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralenDihydrochloride (Compound 8)

The synthesis of4'(12-amino-8-aza-2,5-dioxa)dodecyl-4,5',8-trimethylpsoralendihydrochloride proceeds in one (1) step from the product of Example 3,step 2: A solution of4'-(7-methanesulfonyloxy-2,5-oxa)heptyl-4,5',8-trimethylpsoralen (108mg, 0.253 mmol) in 8 mL of acetonitrile was slowly added to a solutionof 1, 4-diaminobutane (132 mg, 1.49 mmol) in 2.8 mL of acetonitrile.After refluxing for 8 hours, no starting material remained by TLC. Thereaction mixture was cooled to room temperature and CHCl₃ (25 mL) and 1Naqueous NaOH (25 mL) solution were added. The layers were separated andCHCl₃ (2×10 mL) was used to wash the aqueous layer. Aqueous HCl (0.3N,3×10 mL) was used to extract the product from the combined organicslayers. The HCl layers was treated with 20% aqueous NaOH solution untilpH 13. The combined basic layers were then extracted with CHCl₃ (3×20mL). The CHCl₃ layer was washed with saturated NaCl aqueous solution (10mL) then dried over anhydrous Na₂ SO₄. After concentration and vacuumdrying, 63 mg of product was obtained (60%). NMR δ1.45 (m, 2H), 2.49 (s,6H),2.55 (s, 3H),2.58 (t, 2H),2.66 (t, J=5.6 Hz, 2H),2.76 (m, 4H),3.55-3.61 (m, 6H), 4.68 (s, 2H),6.22 (s, 1H),7.61 (s, 1H).

EXAMPLE 5 Synthesis of 4'-(2-aminoethyl)-4,5',8-trimethylpsoralenHydrochloride (Compound 3)

The synthesis of 4'-(2-aminoethyl)-4,5',8-trimethylpsoralen proceeds inone (1) step: sodium trifluoroacetoxyborohydride was made by addingtrifluoroacetic acid (296 mg, 2.60 mmol) in 2 mL of THF to a stirredsuspension of sodium borohydride (175 mg, 4.63 mmol) in 2 mL of THF overa period of 10 minutes at room temperature. The resultant suspension wasadded to a suspension of 4'-cyanomethyl-4,5',8-trimethylpsoralen(Kaufman et al., J. Heterocyclic Chem. 19:1051 (1982)) (188 mg, 0.703mmol) in 2 mL of THF. The mixture was stirred overnight at roomtemperature. Several drops of water were added to the reacted lightyellow clear solution to decompose the excess reagent under 10° C. Theresulting mixture was concentrated and 1N aqueous NaOH solution (30mL)was added. Chloroform (30 mL then 10 mL, 5 mL)) was used to extract theresultant amine. Combined CHCl₃ layers were washed with saturated NaClsolution. The amine was then extracted into aqueous 0.3N HCl (10, 5, 5mL) and the acid layers were taken to pH 13 with 20% aqueous NaOH. CHCl₃(3×10 mL) was used to extract the amine from the combined base layersthen washed with water (2 mL) and dried over anhydrous Na₂ SO₄. Uponconcentration and vacuum drying the amine was obtained as a solid, >95%pure by NMR. NMR δ2.45 (s, 3H); 2.47 (s, 3H); 2.53 (s, 3H); 2.78 (t,J=6.6 Hz, 2H); 3.00 (t, J=6.5 Hz, 2H); 6.20 (s, 1H); 7.44 (s, 1H). Thesolid was dissolved in absolute ethanol. A solution of hydrogen chloridein diethyl ether (1N, 1 mL) was added. The suspension was filtered toobtain compound 3, a light purple solid (32.7 mg, yield 15%), m.p. >237°C. (decomp.)

EXAMPLE 6 4'-(6-Aminohexyl-2-aza)-4,5',8-trimethylpsoralenDihydrochloride (Compound 6)

The synthesis of 4'-(6-aminohexyl-2-aza)-4,5',8-trimethylpsoralendihydrochloride proceeds in one (1) step, as follows: a solution of4'-chloromethyl-4,5',8-trimethylpsoralen (188 mg, 0.68 mmol) in 30 mL ofacetonitrile was added to a solution of 1,4-diaminobutane (120 mg, 1.4mmol) in 7 mL of acetonitrile. After stirring overnight the solvent wasremoved under reduced pressure. Chloroform (10 mL) and 1N NaOH (10 mL)were added to the residue and the mixture was shaken and separated. Theaqueous solution was extracted with a further 2×10 mL of CHCl₃ and thecombined extracts were rinsed with water. The product was then extractedfrom the CHCl₃ solution with 0.3N aqueous HCl and the acidic layer wasthen taken to pH 12 with concentrated NaOH solution. The base suspensionwas extracted with CHCl₃ which was then rinsed with water, dried overNa₂ SO₄ and concentrated under reduced pressure to give the amine as thefree base; NMR (CDCl3); δ1.33 (m, 3H), 1.52 (m, 4H),2.47 (s, 3H),2.49(d, J=1.1 Hz, 3H),2.54 (s, 3H),2.68 (q, J=6.5 Hz, 4H),3.86 (s, 2H), 6.21(apparent d, J=1.1 Hz, 1H),7.60 (s, 1H).

The free base, dissolved in about 6 mL of absolute EtOH was treated witha solution of HCl in ether (1.0M, 3 mL). The resultant HCl salt wasfiltered, rinsed with absolute EtOH and dried under vacuum to yield 150mg of compound 6, (55%), m.p. 290° C. (decomposed). Analysis calculatedfor C₁₉ H₂₆ C₁₂ N₂ O₃.H₂ O: C,54.42; H, 6.73; N, 6.68. Found: C, 54.08;H, 6.45; N, 6.65.

Similarly prepared were:

a) 4'-(4-amino-2-aza)butyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 1), mp 320°-322° C. (decomp).

b) 4'-(5-amino-2-aza)pentyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 5), mp 288° C. (decomp). NMR of free base: d 1.33 (br s, 3H),1.66 (pent, J=6.8 Hz, 2H), 2.47 (s, 3H),2.50 (d, J=1 Hz, 3H),2.55 (s,3H), 2.6-2.85 (m, 4H), 3.89 (s, 2H),6.22 (apparent d, J=1 Hz, 1H), 7.62(s, 1H).

c) 4'-(7-amino-2-aza)heptyl-4,5',8-trimethylpsoralen dihydrochloride(Compound 10), mp 300° C. (decomp). NMR of free base: d 1.22 (brs,),1.3-1.6 (m) total 9H, 2.44 (s),2.50 (s), total 9H, 2.63 (m, 4H),6.17(s, 1H),7.56 (s, 1H).

EXAMPLE 7 5'-(6-Amino-2-aza)hexyl-4,4',8-trimethylpsoralenDihydrochloride (Compound 17)

The synthesis of 5'-(6-amino-2-aza)hexyl-4,4',8-trimethylpsoralendihydrochloride proceeds in one (1) step, as follows: a suspension of5'-chloromethyl-4,4',8-trimethylpsoralen (190 mg, 0.68 mmol) in 30 mL ofacetonitrile was added to a solution of 1,4-diaminobutane (120 mg, 1.4mmol) in 7 mL of acetonitrile. After stirring at room temperatureovernight, the solvent was removed under reduced pressure. Chloroform(10 mL) and 1N NaOH (10 mL) were added to the residue and the mixturewas shaken and separated. The aqueous layer was extracted with a further2×10 mL of CHCl₃ and the combined extracts were rinsed with water. Theproduct was then extracted from the CHCl₃ solution with 0.3N aqueous HCland the acidic layer was then taken to approximately pH 12 withconcentrated NaOH solution. The base suspension was extracted with CHCl₃which was then rinsed with water, dried over Na₂ SO₄ and concentratedunder reduced pressure.

The residue was purified by column chromatography on silica gel withCHCl₃ : EtOH: Et₃ N (9:1:0.25). The fractions containing the productwere combined and stripped of the solvent to give the free amine. NMR(CDCl₃): δ1.35 (m, 3H); 1.49 (m, 4H); 2.22 (s, 3H); 2.46 (d, J=1.1 Hz,3H); 2.51 (S, 3H); 2.65 (m, 4H); 3.88 (s, 2H); 6.17 (apparent d, 1 Hz);7.40 (s, 1H).

The free base, dissolved in absolute EtOH (˜6 mL) was treated with asolution of HCl in ether (1.0M,˜3 mL). The resultant HCl salt wasfiltered, rinsed with absolute EtOH and dried under vacuum to yield 100mg (36.3%) of product, m.p. 288° C. (decomposed).

Similarly prepared was 5'-(4-amino-2-aza)butyl-4,4',8-trimethylpsoralendihydrochloride (Compound 16). NMR of free base: δ1.83 (br s, 3H),2.27(s, 3H), 2.51 (s, 3H),2.58 (s, 3H),2.74 (m, 2H),2.87 (m, 2H),3.95 (s,2H),6.24 (s, 1H), 7.46 (s, 1H).

EXAMPLE 8 4'-(14-Amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralenTetrahydrochloride (Compound 15)

The synthesis of 4'-(14-amino-2,6,11-triaza)tetradecyl-4,5',8-trimethylpsoralen tetrahydrochloride proceeds in one(1) step, as follows. To a solution of 0.5 g (2.5 mmol) of spermine(Aldrich, Milwaukee, Wis.) in 10 ml of methanol was added a 5Nmethanolic solution of HCl (concentrated HCl diluted with MeOH to 5N) toadjust to pH 5-6, followed by 0.128 g (0.5 mmol) of4,5',8-trimethylpsoralen-4'carboxaldehyde, 20 mg (0.3 mmol) of NaBH₃ CNand 3 mL of MeOH. The reaction mixture was stirred at room temperatureovernight. A solution of 5N methanolic HCl was added until pH<2 andmethanol was removed under reduced pressure. The residue was taken up inabout 100 mL of water and rinsed with three 25 mL portions of CHCl₃. Theaqueous solution was brought to pH>10 with concentrated NaOH andextracted with three 25 mL portions of CHCl₃. These final extracts werecombined and washed with water, dried (Na₂ SO₄ ) and evaporated to givethe free base of the amine, ≧95% pure by NMR. NMR (CDCl₃): d 1.31 (m,5H),1.45 (pent, J=3.41 Hz, 4H),1.65 (m, 4H), 2.46 (s, 3H),2.49 (d,J=1.14 Hz, 3H), 2.66 (m, 15H),3.85 (s, 2H),6.21 (s, 1H)m 7.60 (s, 1H).

The free amine was dissolved in absolute ethanol and HCl (anhydrous, 1Nin ethyl ether) was added. The hydrochloride salt was filtered andwashed with absolute ethanol and dried under vacuum at room temperaturegiving 80.2 mg of product as a light yellow solid.

EXAMPLE 9

The assay used to predict pathogen inactivation efficiency and todetermine nucleic acid binding of the photoreactive binding compounds ofthe present invention was that in which a bacteriophage, R17, insolution with the desired substrate was irradiated. The ability of thephage to subsequently infect bacteria and inhibit their growth wasmeasured. The bacteriophage was selected for its relatively accessiblenucleic acid such that the culture growth inhibition would accuratelyreflect nucleic acid damage by the test compounds. The bacteriophageassay for nucleic acid binding to test compounds offers a safe andinexpensive procedure to identify compounds likely to display efficientpathogen inactivation. The phages have also been shown to accuratelyreflect HIV-1 sensitivity to similar compounds.

The R17 was grown up in Hfr 3000 bacteria, approximate titer 5×10¹¹.(R17 and Hfr 3000 were obtained from American Tissue Culture Collection(ATCC), Washington, D.C.) The R17 phage stock was added to a solution of15% fetal bovine serum in DMEM to a final phage concentration of 10⁹/mL. An aliquot (0.5 mL) was transferred to a 1.5 mL snap-toppolyethylene tube. An aliquot (0.004-0.040 mL) of the test compoundstock solution prepared in water, ethanol or dimethylsulfoxide at0.80-8.0 mM was added to the tube. Compounds were tested atconcentrations between 4 μM and 320 μM. (AMT is commercially availablefrom HRI, Inc., Concord, Calif. 8-MOP is commercially available fromSigma, St. Louis, Mo.). The tubes were placed in a light device asdescribed in EXAMPLE 1 and irradiated for between 1 and 10 minutes.Sterile 13 mL dilution tubes were prepared; each test compound requiredone tube with 0.4 mL of LB broth and five tubes containing 0.5 mL of LBbroth. To make the dilutions, a 0.100 mL aliquot of the irradiatedsolution of phage and test compound was added to the first dilution tubeof 0.4 mL of media then 0.020 mL of this solution was added to thesecond tube of 0.5 mL medium (1:25). The second solution was thendiluted serially (1:25) into the remaining tubes. To each diluted samplewas added 0.050 mL of Hfr 3000 bacteria cultured overnight and 3 mL ofmolten LB top agar and the mixed materials were poured onto LB brothplates. After the top agar hardened, the plates were incubated at 37° C.overnight. The plaque forming units were then counted the followingmorning and the titer of the phage remaining after phototreatment wascalculated based on the dilution factors.

The following controls were run: the "phage only" in which phage was nottreated with test compound and not irradiated (listed as "startingtiter" in the tables below); the "UV only" in which the phage wasirradiated in the absence of test compound; and the "dark" control inwhich the phage/test compound solution was not irradiated before it wasdiluted and plated.

TABLE 3, below, shows three different experiments which tested Compound1 according to the R17 protocol just described. A comparison of valuesfor the control samples in runs 1-3 (values in bold) shows that neitherthe "UV only" nor the "dark" controls result in significant bacterialkill (at most, 0.3 logs killed in the "UV only" control and 0.1 logskilled in the "dark" control).

The "UV only" control was repeated in many similar experiments withother compounds of the present invention and consistently showed nosignificant kill. (Data not shown). Thus, the "UV only" control is notshown in the tables and figures that follow, although it was performedin every experiment in this example. As for the "dark" control, aftermany trials with various compounds of the present invention, it becameapparent that regardless of the type of substitution on the 4' positionof the psoralen, no experimentally significant bacterial inactivationwas observed in the dark. (Data not shown). For example, in Table 3,experiment 1 shows 0.1 logs kill with compound 1 in the dark. Incontrast, when Compound 1 is irradiated for just 1 minute, the resultingdrop in titer is >6.7 logs. Therefor, "dark" controls were not run forthe later tested compounds and where run, are not shown in the tablesand figures that follow.

Tables 4-7, below, and FIGS. 6-8 show the results of the R17 assay forseveral of the 4'-primaryamino-substituted psoralen compounds of thepresent invention. The data in Tables 5 and 6 appears in FIGS. 6 and 7,respectively. 5'-Primaryamino-substituted psoralen compounds of thepresent invention, which have substitutions on the 5' position similarto the 4'-primaryamino-substituted psoralen compounds, were also testedat varying concentration, as described above in this example, and areshown to exhibit comparable inactivation efficiency. The results forthese compounds are shown in FIGS. 9 and 10, below.

                  TABLE 3                                                         ______________________________________                                                                 LOG        LOGS                                      EXPERIMENT # TREATMENT   TITER      KILLED                                    ______________________________________                                        1            phage only  7.7        --                                                     uva only (10')                                                                            7.4        0.3                                                    compound only                                                                             7.6        0.1                                                    (32 μM)                                                                    32 μM cmpd                                                                             <1         >6.7                                                   1' uva                                                                        32 μM cmpd                                                                             <1         >6.7                                                   10' uva                                                          2            phage only  7.8        --                                                     uva only (10')                                                                            7.6        0.2                                                    compound only                                                                             7.7        0.1                                                    (3.2 μM)                                                                   3.2 μM cmpd                                                                            6.9        0.9                                                    1' uva                                                                        3.2 μM cmpd                                                                            6.1        1.7                                                    10' uva                                                          3            phage only  7.3        --                                                     uva only (1')                                                                             7.3        0                                                      compound only                                                                             7.3        0                                                      (16 μM)                                                                    4 μM cmpd                                                                              6.3        1.0                                                    1' uva                                                                        8 μM cmpd                                                                              5.6        1.7                                                    1' uva                                                                        16 μM cmpd                                                                             3.9        3.4                                                    1' uva                                                           ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Starting titer of R17: approx. 7.5 logs                                       1 Minute Irradiation                                                                                         R17 log kill                                   Cmpd.   Structure              (32 μM)                                     ______________________________________                                        AMT                                                                                    ##STR8##              >6.7                                           8-MOP                                                                                  ##STR9##              0                                                       ##STR10##             >6.6                                           ______________________________________                                    

                                      TABLE 5                                     __________________________________________________________________________    Starting titer approx. 7.2 logs R17                                           1 minute irradiation                                                                                 R17 log kill                                           Compound                                                                            Structure        8 uM                                                                              16 uM 32 uM                                        __________________________________________________________________________    AMT                                                                                  ##STR11##       2.7 4.6   >6.2                                                ##STR12##       1.7 2.8    5.3                                         2                                                                                    ##STR13##       3.8 >6.2  >6.2                                         3                                                                                    ##STR14##       >6.2                                                                              >6.2  >6.2                                         __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Starting titer approx. 7.1 logs                                               1 minute irradiation = 1.2 J/cm.sup.2                                                              R17 log kill                                             Cmpd.                                                                             Structure        8 uM                                                                              16 uM                                                                             32 uM                                                                             64 uM                                        __________________________________________________________________________    AMT                                                                                ##STR15##       --  4.5 4.8 --                                                ##STR16##       5.6 >6.1                                                                              --  --                                           4                                                                                  ##STR17##       --  2.3 4.3 >6.1                                         5                                                                                  ##STR18##       --  5.6 >6.1                                                                              >6.1                                         6                                                                                  ##STR19##       --  >6.1                                                                              >6.1                                                                              >6.1                                         __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Starting titer approx. 7.1 logs R17.                                          1 Minute Irradiation.                                                                              R17 log kill                                             Cmpd.                                                                             Structure        8 uM                                                                              16 uM                                                                             32 uM                                                                             64 uM                                        __________________________________________________________________________    AMT                                                                                ##STR20##       --  >6  >6  --                                                ##STR21##       >6  >6  --  --                                           7                                                                                  ##STR22##       --  >6  >6  >6                                           __________________________________________________________________________

The compounds of the present invention having substitutions on the 4'position of the psoralen ring proved to be active in killing R17, asshown in the tables above. In Table 4, it is apparent that compound 1 ofthe present invention exhibits much higher R17 inactivation efficiencythan does 8-MOP. As shown in Table 5 and FIG. 6, Compound 1 is one ofthe less active compounds of the present invention. Both Compounds 2 and3 show higher log inactivation than Compound 1 at each concentrationpoint. These results support that the compounds of the present inventionare generally much more active than 8-MOP.

The compounds of the present invention also have similar or better R17inactivation efficiency than AMT. In Tables 5 and 6, and FIGS. 6-10, allcompounds of the present invention achieve R17 log inactivation atlevels comparable to AMT. Compounds 2 and 3 (Table 5, FIG. 6), Compounds5 and 6 (Table 6, FIG. 7), and Compound 16 (FIG. 10) exhibitsignificantly higher inactivation efficiency than does AMT.

Compounds of the present invention were also tested at a constantconcentration for varying doses of UV light. Three sets of 1.5 mL tubeswere prepared containing 0.6 mL aliquots of R17 in DMEM (prepared asdescribed above). The compound tested was added at the desiredconcentration and the samples were vortexed. The samples were thenirradiated at intervals of 1.0 J/cm², until 3.0 J/cm² was reached.Between each 1.0 J/cm² interval, 100 μL was removed from each sample andplaced in the first corresponding dilution tube, then five sequentialdilutions were performed for each compound tested, at all 3 irradiationdoses, as described above in this example.

Then 50 μL of Hfr 3000 bacteria was added to each tube, 3 mL of top agarwas added and the tube contents were vortexed. The contents of each tubewas poured into its own LB plate and the plates were incubated overnightat 37° C. Plaques were counted by visual inspection the followingmorning.

The results of the assay for several 4' and 5' -primaryamino-substitutedpsoralen compounds are shown in FIGS. 11-17. This data further supportsthat the compounds of the present invention are comparable to AMT intheir ability to inactivate R17. Further, Compounds 6 (FIG. 11), 10(FIG. 12), 12 (FIG. 13), 15 (FIG. 14 and 17), and Compound 17 (FIG. 15),all were more efficient at inactivating R17 than was AMT.

EXAMPLE 10

Pathogen inactivation efficiency of several compounds of the presentinvention was evaluated by examining the ability of the compounds toinactivate cell-free virus (HIV). Inactivation of cell-free HIV wasperformed as follows.

As in the R17 assay, small aliquots of the compounds listed in TABLES 8and 9, below, at the concentrations listed in the table, were added tostock HIV-1 to a total of 0.5 mL. The stock HIV (10⁵ -10⁷ plaque formingunits/mL) was in DMEM/15% FBS. The 0.5 mL test aliquots were placed in24 well polystyrene tissue culture plates and irradiated with 320-400 nm(20 mW/cm²) for 1 min on a device similar to the device of Example 1.The photoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Controls included HIV-1 stock only, HIV-1 plus UVA only, andHIV-1 plus the highest concentration of each psoralen tested, with noUVA. Post irradiation, all samples were stores frozen at -70° C. untilassayed for infectivity by a microtiter plaque assay. Aliquots formeasurement of residual HIV infectivity in the samples treated with acompound of the present invention were withdrawn and cultured.

Residual HIV infectivity was assayed using an MT-2 infectivity assay.(Previously described in Hanson, C. V., Crowford-Miksza, L. andSheppard, H. W., J. Clin. Micro 28:2030 (1990)). The assay medium was85% DMEM (with a high glucose concentration) containing 100 μg ofstreptomycin, 100 U of penicillin, 50 μg of gentamicin, and 1 μg ofamphotericin B per mL, 15% FBS and 2 μg of Polybrene (Sigma ChemicalCo., St. Louis, Mo.) per mL. Test and control samples from theinactivation procedure were diluted in 50% assay medium and 50% normalhuman pooled plasma. The samples were serially diluted directly in96-well plates (Corning Glass Works, Corning, N.Y.). The plates weremixed on an oscillatory shaker for 30 seconds and incubated at 37° C. ina 5% CO₂ atmosphere for 1 to 18 hours. MT-2 cells (0.025 mL) [clonealpha-4, available (catalog number 237) from the National Institutes ofHealth AIDS Research and Reference Reagent Program, Rockville, Md.] wereadded to each well to give a concentration of 80,000 cells per well.After an additional 1 hour of incubation at 37° C. in 5% CO₂, 0.075 mLof assay medium containing 1.6% SeaPlaque agarose (FMC Bioproducts,Rockland, Me.) and prewarmed to 38.5° C. was added to each well. Theplates were kept at 37° C. for a few minutes until several plates hadaccumulated and then centrifuged in plate carriers at 600×g for 20minutes in a centrifuge precooled to 10° C. In the centrifuge, cellmonolayers formed prior to gelling of the agarose layer. The plates wereincubated for 5 days at 37° C. in 5% CO₂ and stained by the addition of0.05 mL of 50 μg/mL propidium iodide (Sigma Chemical Co.) inphosphate-buffered saline (pH 7.4) to each well. After 24 to 48 hours,the red fluorescence-stained microplaques were visualized by placing theplates on an 8,000 μW/cm² 304 nm UV light box (Fotodyne, Inc., NewBerlin, Wis.). The plaques were counted at a magnification of ×20 to ×25through a stereomicroscope. The results are shown in TABLES 8 and 9,below. "n" represents the number of runs for which the data point is anaverage.

The results support that the compounds of the present invention areeffective in inactivating HIV. In fact, the data for concentrations of64 μM of compound or higher suggests that compounds 2 and 3 aresignificantly more active than AMT, which was previously thought to beone of the most active anti-viral psoralens. At lower concentrations,Compound 6 is able to kill a higher log of HIV (3.1 logs at 32 μM) thanis AMT (2.5 logs at 32 μM). The other compounds listed in TABLE 8display inactivation efficiency in the same range as AMT.

                  TABLE 8                                                         ______________________________________                                        1 minute irradiation                                                          HIV starting titer: approximately 5 logs                                               HIV log kill                                                         COMPOUND   16 μM                                                                              32 μM  64 μM                                                                              128 μM                                ______________________________________                                        AMT        1.4     1.9->3.6  3.9->3.6                                                                              >4.1                                     1          --      --         2.1    >2.8                                     2          1.4     3.8       >4.5    >4.5                                     3          --      2.7       >3.8    >3.8                                     4          --      2.2       >3.6    >3.6                                     5          0.9     1.3       >2.6    --                                       6          2.0     3.1       >3.8    --                                       7          0.8     2.1        3.5    --                                       8          1.1     1.9        3.7    >3.7                                     ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        HIV starting titer: approximately 5.4 logs                                    1 minute irradiation                                                                     HIV log kill                                                       COMPOUND     16 μM    32 μM                                                                              64 μM                                     ______________________________________                                         6           2.1         3.2     >2.8                                          9           0.8         1.4     2.7                                          10           2.0         >3.5    >3.5                                         12           0.4         0.8     1.3                                          17           1.2         2.9     3.4                                          18           1.0         1.0     3.1                                          ______________________________________                                    

EXAMPLE 11

This example describes the protocol for inactivation of another virus,Duck Hepatitis B Virus (DHBV), using compounds of the present invention.

DHBV in duck yolk was added to platelet concentrate (PC) to a finalconcentration of 2×107 particles per mL and mixed by gentle rocking for≧15 min. Psoralens S-70, S-59 and AMT were added to 3 mL aliquots of PCin a Teflon™ mini-bag at concentrations of 35, 70, and 100 mM. Samples,including controls without added psoralen, were irradiated with 5J/CM²UVA, with mixing at 1 J/cm² increments. After irradiation, leukocytesand platelets were separated from virus by centrifugation. Thesupernatant containing DHBV was digested overnight with 50 μg/mLproteinase K in a buffer containing 0.5% sodium dodecyl sulphate, 20 mMTris buffer, pH 8.0, and 5 mM EDTA at 55° C. Samples were extracted withphenol-chloroform and chloroform, followed by ethanol precipitation.Purified DNA was then used in PCR amplification reactions with astarting input of 10⁶ DHBV genomes from each sample. PCR amplicons weregenerated using primers pairs DCD03/DCD05 (127 bp), DCD03/DCD06 (327 bp)and DCD03/DCD07 (1072 bp). PCR was performed in a standard PCR buffercontaining 0.2 mM each deoxyribonucleoside 5'-triphosphates (dATP, dGTP,dCTP, and dTTP), 0.5 mM each primer, and 0.5 units Taq polymerase per100 ml reaction. 30 cycles of amplification were performed with thefollowing thermal profile: 95° C. 30 sec, 60° C. 30 sec, 72° C. min. Theamplification was followed by a 7 min incubation at 72° C. to yield fulllength products. [lambda-³² P] dCTP was added at an amount of 10 mCi per100 ml in order to detect and quantify the resulting products. Productswere separated by electrophoresis on denaturing polyacrylamide slab gelsand counted. The absence of signal in a given reaction was taken toindicate effective inactivation of DHBV.

The results showed that the smaller amplicons displayed increasinginactivation as a function of psoralen concentration for all psoralenstested. At the same concentrations, S-59 and S-70 inhibited PCR of thesmaller amplicons better than did AMT. For the 1072 bp amplicon,complete inhibition of PCR was observed at all concentrations of S-59and S-70, whereas the sample without psoralen gave a strong signal. AMTinhibited PCR amplification of the 1072 bp amplicon at the 70 and 100 mMlevels, but a signal could be detected when AMT was used at 35 mM finalconcentration.

EXAMPLE 12

In Example 10, the compounds of the present invention were tested fortheir ability to inactivate virus in DMEM/15% FBS. In this example, thecompounds are tested in both 100% plasma and predominantly syntheticmedia, to show that the methods of the present invention are notrestricted to any particular type of medium.

For the samples in synthetic media: standard human platelet concentrateswere centrifuged to separate plasma. Eighty-five percent of the plasmawas then expressed off and replaced with a synthetic medium (referred toas "Sterilyte™ 3.0") containing 20 mM Na acetate, 2 mM glucose, 4 mMKCl, 100 mM NaCl, 10 mM Na₃ Citrate, 20 mM NaH₂ PO₄ /Na₂ HPO₄, and 2 mMMgC₂. H9 cells infected with HIV were added to either the 85% Sterilyte™3.0 platelet concentrates or standard human platelet concentrates(2.5×10⁷ cells per concentrate), final concentration 5×10⁵ cells /mL.The platelet concentrates were placed in Teflon™ modified FL20 orTeflon^(TM) Minibags (American Fluoroseal Co., Silver Springs, Md.),treated with one of the compounds shown in FIGS. 18 and 19, at theconcentrations shown, and then irradiated with 320-400 nm (20 mW/cm2)for 5 J/cm² (for plasma samples) or 2 J/cm² (for 85% Sterilyte™ 3.0samples) on a device similar to the Device of Example 1. Thephotoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Aliquots for measurement of residual HIV infectivity in thesamples treated with a compound of the present invention were withdrawnand cultured.

For samples run in plasma: H9 cells infected with HIV were added tostandard human platelet concentrates (2.5×10⁷ cells per concentrate),final concentration 5×10⁵ cells/mL. Aliquots of HIV contaminatedplatelet concentrate (5 mL) were placed in water jacketed Pyrexchambers. The chambers had previously been coated on the inside withsilicon. The platelet concentrates were treated with one of thecompounds listed in TABLES 8 and 9, below, at the concentrations listedin the table, and then irradiated with 320-400 nm (20 mW/cm2) for 1minute on a device similar to the Device of Example 1. Thephotoactivation device used here was previously tested and found toresult in light exposure comparable to the Device of Example 1. (Datanot shown). Aliquots for measurement of residual HIV infectivity in thesamples treated with a compound of the present invention were withdrawnand cultured. Residual HIV infectivity was assayed for both the plasmaand the 85% Sterilyte™ samples using an MT-2 infectivity assay.(Detailed in Example 10, above, and previously described in Hanson, C.V., et al., J. Clin. Micro 28:2030 (1990)). The results are shown inFIGS. 18 and 19.

The results support that the compounds of the present invention areeffective in inactivating HIV in both plasma and synthetic medium.Comparing FIGS. 18 and 19, the inactivation curves appear to be thesame, both achieving approximately 5 logs of inactivation at 64 μMconcentrations of compound. However, the inactivation in synthetic mediawas performed with only 2 J/cm² irradiation, 3 J/cm² less than thatrequired to achieve the same inactivation in plasma. Thus, it appearsfrom the data that synthetic media facilitates the inactivation methodsof the present invention.

EXAMPLE 13

In this example bacterial inactivation by the photoreactive nucleic acidbinding compounds of the present invention was measured as a function ofthe ability of the bacteria to subsequently replicate. A gram negativebacteria was chosen as representative of the more difficult bacterialstrains to inactivate.

The bacteria, a strain of Pseudomonus, was innoculated into LB with asterile loop and grown overnight in a shaker at 37° C. Based on theapproximation that one OD at 610 nm is equivalent to 5×10⁸ colonyforming units (cfu)/mL, a 1:10 dilution of the culture was measured on aspectrophotometer, (manufactured by Shimatsu). The bacterial culture wasadded to a solution of 15% fetal bovine serum in DMEM to a finalbacteria concentration of approximately 10⁶ /mL. An aliquot (0.8 mL) wastransferred to a 1.5 mL snap-top polyethylene tube. An aliquot(0.004-0.040 mL) of the test compound stock solution prepared in water,ethanol or dimethylsulfoxide at 0.80-8.0 mM was added to the tube.Compounds were tested at a concentration of 16 μM. The tubes were placedin a light device as described in EXAMPLE 1 and irradiated with 1.3J/cm², 1.2 J/ cm², and finally 2.5 J/cm², for a total of 5 J/cm.sup. 2.150 μL were removed for testing after each pulse period. Sterile 13 mLdilution tubes were prepared; each test compound required one tube with0.4 mL of LB broth and four tubes containing 0.5 mL of LB broth. To makethe dilutions, a 0.050 mL aliquot of the irradiated solution of phageand test compound was added to the first dilution tube of 0.5 mL ofmedia then 0.050 mL of this solution was added to the second tube of 0.5mL medium (1:10). The second solution was then diluted serially (1:10)into the remaining tubes. 100 μL of the original sample and eachdilution are plated seperately onto LB agar plates and incubated at 37°C. overnight. The colony forming units were then counted the followingmorning and the titer of the phage remaining after phototreatment wascalculated based on the dilution factors.

The following controls were run: the "bacteria only" in which bacteriawas not treated with test compound and not irradiated (listed as"starting titer" in the tables below); the "UV only" in which thebacteria was irradiated in the absence of test compound. Dark controlswere not performed here for reasons set forth in Example 9 above.

The results were as follows. The starting titer of bacteria was 6.5logs. After 5 J/cm² irradiation, the log kill for the various compoundstested were as follows: 8-MOP--1.9 logs, AMT--5.2 logs, Compound2-->5.5, Compound 6-->5.5. From these results, it is clear that thecompounds of the present invention are more efficient than both AMT and8-MOP at inactivating a gram negative bacteria.

EXAMPLE 14

In the above examples, psoralens of the present invention have beendemonstratated to be effective for inactivating pathogens, such asbacteria (pseudomonus), bacteriophage (R17 ) and viruses (HIV and DHBV).Without intending to be limited to any method by which the compounds ofthe present invention inactivate pathogens, it is believed thatinactivation results from light induced binding of the psoralens to thenucleic acid of the pathogens. As discussed above, AMT is known both forits pathogen inactivation efficiency and its accompanying mutagenicaction in the dark at low concentrations. In contrast, the less activepsoralens, such as 8-MOP, that have been examined previously, showsignificantly less mutagenicity. This example establishes thatphotobinding and mutagenicity are not linked phenomenon in the compoundsof the present invention. The psoralens of the present invention haveexceptional pathogen inactivation efficiency while displaying onlyminimal mutagenicity.

In this example the compounds of the present invention are tested fortheir dark mutagenicity using an Ames assay. The procedures used for theSalmonella mutagenicity test as described in detail by Maron and Ameswere followed exactly. Maron, D. M. and B. N. Ames, Mutation Research113:173 (1983). A brief description for each procedure is given here.The tester strains TA97a, TA98, TA100, TA102, TA1537 and TA1538 wereobtained from Dr. Ames. TA97a, TA98, TA1537 and TA1538 are frameshifttester strains. TA100 and TA102 are base-substitution tester strains.Upon receipt each strain was cultured under a variety of conditions toconfirm the genotypes specific to the strains.

The standard Salmonella tester strains used in this study requirehistidine for growth since each tester strain contains a different typeof mutation in the histidine operon. In addition to the histidinemutation, these tester strains contain other mutations, described below,that greatly increase their ability to detect mutagen.

Histidine Dependence: The requirement for histidine was tested bystreaking each strain first on a minimal glucose plate supplemented onlywith biotin and then on a minimal glucose plate supplemented with biotinand histidine. All strains grew the lack of growth of the strains in theabsence of histidine.

rfa Mutation: A mutation which causes partial loss of thelipopolysaccharide barrier that coats the surface of the bacteria thusincreasing permeability to large molecules was confirmed by exposing astreaked nutrient agar plate coated with the tester strain to crystalviolet. First 100 μL of each culture was added to 2 mL of molten minimaltop agar and poured onto a nutrient agar plate. Then a sterile filterpaper disc saturated with crystal violet was placed at the center ofeach plate. After 16 hours of incubation at 37° C. the plates werescored and a clear zone of no bacterial growth was found around thedisc, confirming the rfa mutation.

uvrB Mutation: Three strains used in this study contain a deficient UVrepair system (TA97a, TA98, TA100, TA1537 and TA1538). This trait wastested for by streaking the strains on a nutrient agar plate, coveringhalf of the plate, and irradiating the exposed side of the plate withgermicidal lamps. After incubation growth was only seen on the side ofthe plate shielded from UV irradiation.

R-factor: The tester strains (TA97a, TA98, TA100, and TA102) contain thepKM101 plasmid that increases their sensitivity to mutagens. The plasmidalso confers resistance to ampicillin to the bacteria. This wasconfirmed by growing the strains in the presence of ampicillin.

pAQ1: Strain TA102 also contains the pAQ1 plasmid that further enhancesits sensitivity to mutagens. This plasmid also codes for tetracyclineresistance. To test for the presence of this plasmid TA102 was streakedon a minimal glucose plate containing histidine, biotin, andtetracycline. The plate was incubated for 16 hours at 37° C. The strainshowed normal growth indicating the presence of the pAQ1 plasmid.

The same cultures used for the genotype testing were again cultured andaliquots were frozen under controlled conditions. The cultures wereagain tested for genotype to confirm the fidelity of the genotype uponmanipulation in preparing the frozen permanents.

The first tests done with the strains were to determine the range ofspontaneous reversion for each of the strains. With each mutagenicityexperiment the spontaneous reversion of the tester strains to histidineindependence was measured and expressed as the number of spontaneousrevertants per plate. This served as the background controls. A positivemutagenesis control was included for each tester strain by using adiagnostic mutagen suitable for that strain (2-aminofluorene at 5mg/plate for TA98 and sodium azide at 1.5 mg/plate for TA100).

For all experiments, the pre-incubation procedure was used. In thisprocedure one vial of each tester strain was thawed and 20 μL of thisculture was added to 6 mL of Oxoid Nutrient Broth #2. This solution wasallowed to shake for 10 hours at 37° C. In the pre-incubation procedure,0.1 mL of this overnight culture was added to each of the requirednumber of sterile test tubes. To half of the tubes 0.5 mL of a 10% S-9solution containing Aroclor 1254 induced rat liver extract (MolecularToxicology Inc., Annapolis, Md.), and MgCl₂, KCl, glucose-6-phosphate,NADP, and sodium phosphate buffer (Sigma, St. Louis, Mo.) were added. Tothe other half of the tubes 0.5 mL of 0.2M sodium phospate buffer, pH7.4, was used in place of the S-9 mixture (the -S9 samples). Finally 0.1mL of the test solution containing either 0, 0.1, 0.5, 1, 5, 10, 50,100, 250, or 500 μ g/mL of the test compound was added. The 0.7 mLmixture was vortexed and then pre-incubated while shaking for 20 minutesat 37° C. After shaking, 2 mL of molten top agar supplemented withhistidine and biotin were added to the 0.7 mL mixture and immediatelypoured onto a minimal glucose agar plate (volume of base agar was 20mL). The top agar was allowed 30 minutes to solidify and then the plateswere inverted and incubated for 44 hours at 37° C. After incubation thenumber of revertant colonies on each plate were counted. The resultsappear in TABLES 10 (A)-16 (B), below. ("n" represents the number ofreplicates performed for each data point.)

                                      TABLE 10 (A)                                __________________________________________________________________________    AMT                                                                                TA97a                                                                              TA97a TA98 TA98  TA100 TA100                                        STRAIN                                                                             -S9  +S9   -S9  +S9   -S9   +S9                                          __________________________________________________________________________    Dose                                                                          μg/plate                                                                   0    109  158   20   25    126   123                                               n = 23                                                                             n = 39                                                                              n = 38                                                                             n = 53                                                                              n = 41                                                                              n = 56                                       0.1  14   -23   3    1     -10   -16                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        0.5  9    32    5    3     13    -12                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        1    54   32    5    21    17    -19                                               n = 3                                                                              n = 6 n = 3                                                                              n = 6 n = 3 n = 6                                        5    73   149   16   232   59    -6                                                n = 3                                                                              n = 6 n = 6                                                                              n = 9 n = 9 n = 12                                       10              20   403   105   17                                                           n = 9                                                                              n = 9 n = 15                                                                              n = 15                                       50              69   620   73    52                                                           n = 9                                                                              n = 9 n = 9 n = 9                                        100             114  745   75    85                                                           n = 9                                                                              n = 9 n = 9 n = 9                                        250             112  933   24    89                                                           n = 6                                                                              n = 6 n = 6 n = 6                                        Positive  5 μg/plate                                                                            5 μg/plate                                                                       1.5 μg/plt                                      Control   2-Amino    2-Amino-                                                                            sodium                                                       fluorene   fluorene                                                                            azide                                                        808        1154  965                                                          n = 21     n = 35                                                                              n = 38                                             __________________________________________________________________________

                                      TABLE 10 (B)                                __________________________________________________________________________    AMT                                                                                 TA102 TA102                                                                              TA1537                                                                             TA1537                                                                             TA1538                                                                             TA1538                                        STRAIN                                                                              -S9   +S9  -S9  +S9  -S9  +S9                                           __________________________________________________________________________    Dose                                                                          μg/plate                                                                   0     346   404  9    9    15   19                                                  n = 26                                                                              n = 41                                                                             n = 30                                                                             n = 45                                                                             n = 30                                                                             n = 42                                        0.1   27    -20  0    2    3    3                                                   n = 3 n = 6                                                                              n = 3                                                                              n = 6                                                                              n = 3                                                                              n = 6                                         0.5   47    5    3    2    4    13                                                  n = 3 n = 6                                                                              n = 9                                                                              n = 12                                                                             n = 9                                                                              n = 12                                        1     88    -17  5    3    4    37                                                  n = 3 n = 6                                                                              n = 9                                                                              n = 12                                                                             n = 9                                                                              n = 12                                        5     266   51   44   22   13   177                                                 n = 3 n = 6                                                                              n = 9                                                                              n = 12                                                                             n = 18                                                                             n = 21                                        10               52   30   14   255                                                            n = 9                                                                              n = 9                                                                              n = 9                                                                              n = 9                                         50               2688 94                                                                       n = 9                                                                              n = 9                                                   100              2058 686                                                                      n = 9                                                                              n = 9                                                   250              434  3738                                                                     n = 9                                                                              n = 12                                                  Positive                                                                            100 μg/pl                                                                             10 μg/plt                                                                       10 μg/plt                                                                            5 μg/plate                                 Control                                                                             hydrogen   9-Amino                                                                            2-Amino-  2-Amino-                                            peroxide   acridine                                                                           fluorene  fluorene                                            660        284  73        1064                                                n = 23     n = 6                                                                              n = 24    n = 30                                        __________________________________________________________________________

                  TABLE 11 (A)                                                    ______________________________________                                        8-MOP                                                                                   TA102      TA102    TA1537  TA1537                                  STRAIN    -S9        +S9      -S9     +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0         346        404      9       9                                                 n = 26     n = 41   n = 30  n = 45                                  1         -55        -46                                                                n = 14     n = 17                                                   10        -57        -27                                                                n = 14     n = 17                                                   30                            5       1                                                                     n = 3   n = 6                                   60                            3       1                                                                     n = 3   n = 6                                   90                            -1      -4                                                                    n = 3   n = 6                                   100       217        290                                                                n = 14     n = 17                                                   500       781        1179                                                               n = 11     n = 11                                                   Positive  100 μg/plt       10 μg/plt                                                                          10 μg/plt                            Control   hydrogen            9-Amino-                                                                              2-Amino-                                          peroxide            Acridine                                                                              fluorene                                          660                 284     73                                                n = 23              n =  6  n = 24                                  ______________________________________                                    

                  TABLE 11 (B)                                                    ______________________________________                                        8-MOP                                                                                   TA102      TA102    TA1537  TA1537                                  STRAIN    -S9        +S9      -S9     +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0         346        404      9       9                                                 n = 26     n = 41   n = 30  n = 45                                  1         -55        -46                                                                n = 14     n = 17                                                   10        -57        -27                                                                n = 14     n = 17                                                   30                            5       1                                                                     n = 3   n = 6                                   60                            3       1                                                                     n = 3   n = 6                                   90                            -1      -4                                                                    n = 3   n = 6                                   100       217        290                                                                n = 14     n = 17                                                   500       781        1179                                                               n = 11     n = 11                                                   Positive  100 μg/plt       10 μg/plt                                                                          10 μg/plt                            Control   hydrogen            9-Amino-                                                                              2-Amino-                                          peroxide            Acridine                                                                              fluorene                                          660                 284     73                                                n = 23              n =  6  n = 24                                  ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Compound 1                                                                            TA100       TA100    TA1538  TA1538                                   STRAIN  -S9         +S9      -S9     +S9                                      ______________________________________                                        Dose                                                                          μg/plate                                                                   0       126         123      15      19                                               n = 41      n = 56   n = 30  n = 42                                   5       292         -24      10      21                                               n = 3       n = 3    n = 3   n = 3                                    10      337         -22      12      22                                               n = 3       n = 3    n = 3   n = 3                                    Positive                                                                              1.5 μg/plate              5 μg/plate                            Control Sodium                       2-Amino-                                         Azide                        fluorene                                         965                          1064                                             n = 38                       n = 30                                   ______________________________________                                    

                  TABLE 13 (A)                                                    ______________________________________                                        Compound 2                                                                              TA1537    TA1537    TA1538  TA1538                                  STRAIN    -S9       +S9       -S9     +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0         9         9         15      19                                                n = 30    n = 45    n = 30  n = 42                                  5                             -8      2                                                                     n = 3   n = 3                                   10        36        5         -13     4                                                 n = 3     n = 3     n = 3   n = 3                                   50        282       40                                                                  n = 3     n = 3                                                     100       258       88                                                                  n = 3     n = 3                                                     250       176       744                                                                 n = 3     n = 3                                                     500       114       395                                                                 n = 3     n = 3                                                     Positive  10 μg/plt                                                                            10 μg/plt      5 μg/plate                           Control   9-Amino-  2-Amino-          2-Amino-                                          acridine  fluorene          fluorene                                          284       73                1064                                              n = 6     n = 24            n = 30                                  ______________________________________                                    

                  TABLE 13 (B)                                                    ______________________________________                                        Compound 2                                                                              TA1537    TA1537    TA1538  TA1538                                  STRAIN    -S9       +S9       -S9     +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0         9         9         15      19                                                n = 30    n = 45    n = 30  n = 42                                  5                             -8      2                                                                     n = 3   n = 3                                   10        36        5         -13     4                                                 n = 3     n = 3     n = 3   n = 3                                   50        282       40                                                                  n = 3     n = 3                                                     100       258       88                                                                  n = 3     n = 3                                                     250       176       744                                                                 n = 3     n = 3                                                     500       114       395                                                                 n = 3     n = 3                                                     Positive  10 μg/plt                                                                            10 μg/plt      5 μg/plate                           Control   9-Amino-  2-Amino-          2-Amino-                                          acridine  fluorene          fluorene                                          284       73                1064                                              n = 6     n = 24            n = 30                                  ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Compound 3                                                                              TA100      TA100    TA1538  TA1538                                  STRAIN    -S9        +S9      -S9     +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0         126        123      15      19                                                n = 41     n = 56   n = 30  n = 42                                  5         47         -19      0       1                                                 n = 3      n = 3    n = 3   n = 3                                   10        47         8        -6      9                                                 n = 3      n = 3    n = 3   n = 3                                   Positive  1.5 μg/plt               5 μg/plt                             Control   Sodium                      2-Amino-                                          Azide                       fluorene                                          965                         1064                                              n = 38                      n = 30                                  ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Compound 4                                                                           TA100       TA100    TA1538  TA1538                                    STRAIN -S9         +S9      -S9     +S9                                       ______________________________________                                        Dose                                                                          μg/plate                                                                   0      126         123      15      19                                               n = 41      n = 56   n = 30  n = 42                                    5      -41         -10      -2      7                                                n = 3       n = 3    n = 3   n = 3                                     10     3           -3       -2      -2                                               n = 3       n = 3    n = 3   n = 3                                     Positive                                                                             1.5 μg/plate              5 μg/plate                             Control                                                                              Sodium                       2-Amino-                                         Azide                        fluorene                                         965                          1064                                             n = 38                       n = 30                                    ______________________________________                                    

                  TABLE 16 (A)                                                    ______________________________________                                        Compound 6                                                                            TA98     TA98       TA100     TA100                                   STRAIN  -S9      +S9        -S9       +S9                                     ______________________________________                                        Dose                                                                          μg/plate                                                                   0       20       25         126       123                                             n = 38   n = 53     n = 41    n = 56                                  5                           -32       12                                                                  n = 3     n = 3                                   10      12       -5         3         -5                                              n = 3    n = 3      n = 9     n = 9                                   50      12       2          2         24                                              n = 3    n = 3      n = 6     n = 6                                   100     22       20         -18       -2                                              n = 6    n = 6      n = 6     n = 6                                   250     12       40                   -38                                             n = 3    n = 3                n = 3                                   500     9        52                                                                   n = 3    n = 3                                                        Positive         5 μg/plate                                                                            1.5 μg/plate                                   Control          2-Amino-   Sodium                                                             fluorene   Azide                                                              1154       965                                                                n = 35     n = 38                                            ______________________________________                                    

                  TABLE 16 (B)                                                    ______________________________________                                        Compound 6                                                                           TA1537     TA1537     TA1538  TA1538                                   STRAIN -S9        +S9        -S9     +S9                                      ______________________________________                                        Dose                                                                          μg/plate                                                                   0      9          9          15      19                                              n = 30     n = 45     n = 30  n = 42                                   5                            -5      0                                                                     n = 3   n = 3                                    10     141        -1         -2      8                                               n = 6      n = 6      n = 3   n = 3                                    50     2010       17                                                                 n = 6      n = 6                                                       100    795        35                                                                 n = 6      n = 6                                                       250    228        99                                                                 n = 6      n = 6                                                       500    43         369                                                                n = 3      n = 3                                                       Positive                                                                             10 μg/plate                                                                           10 μg/plate     5 μg/plate                            Control                                                                              9-Amino-   2-Amino-           2-Amino-                                        acridine   fluorene           fluorene                                        284        73                 1064                                            n = 6      n = 24             n = 30                                   ______________________________________                                    

Maron and Ames (1983) describe the conflicting views with regard to thestatistical treatment of data generated from the test. In light of this,this example adopts the simple model of mutagenicity being characterizedby a two-fold or greater increase in the number of revertants abovebackground (in bold in the tables), as well as dose dependent mutagenicresponse to drug.

With regard to 8-MOP, the only mutagenic response detected was a weakbase-substitution mutagen in TA102 at 500 μg/plate (TABLE 14 (B)).

In sharp contrast, AMT (TABLE 13 (A) and 13 (B)) showed frameshiftmutagenicity at between 5 and 10 μg/plate in TA97a and TA98, at 5μg/plate in TA1537 and at 1 μg/plate in TA1538. AMT showed nosignificant base-substitution mutations.

Looking at Compound 1, the only mutagenic response detected was a weakframeshift mutagen in TA1538 at 5 μg/plate in the presence of S9.Compound 1 also displayed mutation in the TA100 strain, but only in theabsence of S9. Compound 2 also showed weak frameshift mutagenicity inthe presence of S9 in TA98 and TA1537. Compounds 3 and 4 showed nomutagenicity. Compound 6 had no base substitution mutagenicity, butshowed a frameshift response in TA98 in the presence of S9 atconcentrations of 250 μg/plate and above. It also showed a response at50 μg/plate in TA1537 in the presence of S9. Both responses aresignificantly below that of AMT, which displayed mutagenicity at muchlower concentrations (5 μg/plate).

From this data it is clear that the compounds of the present inventionare less mutagenic than AMT, as defined by the Ames test. At the sametime, these compounds show much higher inactivation efficiency than8-MOP, as shown in Examples 9 and 13. These two factors support that thecompounds of the present invention combine the best features of both AMTand 8-MOP, high inactivation efficiency and low mutagenicity.

EXAMPLE 15

In Example 12, the compounds of the present invention exhibited theability to inactivate pathogens in synthetic media. This exampledescribes methods by which synthetic media and compounds of the presentinvention may be introduced and used for inactivating pathogens inblood. FIG. 20A schematically shows the standard blood productseparation approach used presently in blood banks. Three bags areintegrated by flexible tubing to create a blood transfer set (200)(e.g., commercially available from Baxter, Deerfield, Ill.). After bloodis drawn into the first bag (201), the entire set is processed bycentrifugation (e.g., Sorvall™ swing bucket centrifuge, Dupont),resulting in packed red cells and platelet rich plasma in the first bag(201). The plasma is expressed off of the first bag (201) (e.g., using aFenwall™ device for plasma expression), through the tubing and into thesecond bag (202). The first bag (201) is then detached and the two bagset is centrifuged to create platelet concentrate and platelet-poorplasma; the latter is expressed off of the second bag (202) into thethird bag (203).

FIG. 20B schematically shows an embodiment of the present invention bywhich synthetic media and photoactivation compound are introduced toplatelet concentrate prepared as in FIG. 20A. A two bag set (300) issterile docked with the platelet concentrate bag (202) (indicated as"P.C."). Sterile docking is well-known to the art. See e.g., U.S. Pat.No. 4,412,835 to D. W. C. Spencer, hereby incorporated by reference. Seealso U.S. Pat. Nos. 4,157,723 and 4,265,280, hereby incorporated byreference. Sterile docking devices are commercially available (e.g.,Terumo, Japan).

One of the bags (301) of the two bag set (300) contains a syntheticmedia formulation of the present invention (indicated as "STERILYTE").In the second step shown in FIG. 20B, the platelet concentrate is mixedwith the synthetic media by transferring the platelet concentrate to thesynthetic media bag (301). The photoactivation compound can be in thebag containing synthetic media (301), added at the point of manufacture.Alternatively, the compound can be mixed with the blood at the point ofcollection, if the compound is added to the blood collection bag (FIG.20A, 201) at the point of manufacture. The compound may be either in dryform or in a solution compatable with the maintainance of blood.

FIG. 20C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 20B. In thisembodiment, platelets have been transferred to a synthetic media bag(301). The photoactivation compound either has already been introducedin the blood collection bag (201) or is present in the synthetic mediabag (301) to which the platelets are now transferred. This bag (301),which has UV light transmission properties and other characteristicssuited for the present invention, is then placed in a device (such asthat described in Example 1, above) and illuminated.

Following phototreatment, the decontaminated platelets are transferredfrom the synthetic media bag (301) into the storage bag (302) of the twobag'set (300). The storage bag can be a commercially available storagebag (e.g., CLX bag from Cutter).

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds, composition, methods, orprocedures shown and described, as modifications and equivalents will beapparent to one skilled in the art.

We claim:
 1. A method of synthesizing4'-(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen, comprising thesteps:a) providing 4'-(ω-hydroxy-2-oxa)alkyl-4,5',8-trimethylpsoralen;b) treating 4'-(ω-hydroxy-2-oxa)alkyl-4,5',8-trimethylpsoralen with abase and methanesulfonyl chloride so that4'-(ω-methanesulfonyloxy-2-oxa)alkyl-4,5',8-trimethylpsoralen isproduced; c) treating4'-(ω-methanesulfonyloxy-2-oxa)alkyl-4,5',8-trimethylpsoralen withsodium azide, so that 4'-(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralenis produced, and d) reducing4'-[(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralen so that4'-[(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen is produced.
 2. Themethod of synthesis described in claim 1, wherein said4'-(ω-amino-2-oxa) alkyl-4,5',8-trimethylpsoralen is4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.
 3. The method ofsynthesis described in claim 2, wherein, in step (d) ,4'-(4-azido-2-oxa) butyl-4,5',8-trimethylpsoralen is reduced withtriphenylphosphine.
 4. A psoralen compound produced by the method ofsynthesis described in claim 1, comprising4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.
 5. A psoralencompound, comprising 4'-(4-amino-2-oxa)butyl -4,5',8-trimethylpsoralen.6. A method of synthesizing 4'-(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen, comprising the steps:a) providing4'-(ω-hydroxy-2-oxa) alkyl-4,5',8-trimethylpsoralen; b) treating4'-(ω-hydroxy-2-oxa) alkyl-4,5',8-trimethylpsoralen with an amine baseand methanesulfonyl chloride so that 4'-(ω-methanesulfonyloxy-2-oxa)alkyl-4,5',8-trimethylpsoralen is produced; c) treating4'-(ω-methanesulfonyloxy-2-oxa) alkyl-4,5',8-trimethylpsoralen withsodium azide, so that 4'-(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralenis produced, and d) reducing 4'-[(ω-azido-2-oxa)alkyl-4,5',8-trimethylpsoralen with triphenylphosphine so that4'-[(ω-amino-2-oxa) alkyl-4,5',8-trimethylpsoralen is produced.
 7. Themethod of synthesis described in claim 6 wherein said 4'-(ω-amino-2-oxa)alkyl-4,5',8-trimethylpsoralen is 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.